Modulation of TCPTP signal transduction by RNA interference

ABSTRACT

Compositions and methods relating to small interfering RNA (siRNA) polynucleotides are provided as pertains to modulation of biological signal transduction. Shown are siRNA polynucleotides that interfere with expression of TCPTP, a member of the protein tyrosine phosphatase (PTP) class of enzymes that mediate signal transduction. In certain preferred embodiments siRNA modulate signal transduction pathways comprising human or murine TCPTP and, in certain further embodiments, insulin receptor, c-jun kinase (JNK) and/or Jak2. Modulation of TCPTP-mediated biological signal transduction has uses in diseases associated with defects in cell proliferation, cell differentiation and/or cell survival, such as metabolic disorders (including diabetes and obesity), cancer, autoimmune disease, infectious and inflammatory disorders and other conditions.

CROSS-REFERENCE TO RELATED APPLICATION

[0001] This application claims the benefit of U.S. Provisional PatentApplication No. 60/383,249 filed May 23, 2002, and U.S. ProvisionalPatent Application No. 60/462,942 filed Apr. 14, 2003, which areincorporated herein by reference in their entirety.

STATEMENT OF GOVERNMENT INTEREST

[0002] The United States government may have certain rights in thisinvention under grant number R01-GM55989 from the National Institutes ofHealth.

BACKGROUND OF THE INVENTION

[0003] 1. Technical Field

[0004] The present invention relates generally to compositions andmethods useful for treating conditions associated with defects in cellproliferation, cell differentiation, and cell survival. The invention ismore particularly related to double-stranded RNA polynucleotides thatinterfere with expression of a protein tyrosine phosphatase, TCPTP, andpolypeptide variants thereof. The present invention is also related tothe use of such RNA polynucleotides to alter activation of signaltransduction pathway components or to alter cellular metabolic processesthat lead to proliferative responses, cell differentiation anddevelopment, and cell survival.

[0005] 2. Description of the Related Art

[0006] Reversible protein tyrosine phosphorylation, coordinated by theaction of protein tyrosine kinases (PTKs) that phosphorylate certaintyrosine residues in polypeptides, and protein tyrosine phosphatases(PTPS) that dephosphorylate certain phosphotyrosine residues, is a keymechanism in regulating many cellular activities. It is becomingapparent that the diversity and complexity of the PTPs and PTKs arecomparable, and that PTPs are equally important in delivering bothpositive and negative signals for proper function of cellular machinery.Regulated tyrosine phosphorylation contributes to specific pathways forbiological signal transduction, including those associated with celldivision, cell survival, apoptosis, proliferation and differentiation.Defects and/or malfunctions in these pathways may underlie certaindisease conditions for which effective means for intervention remainelusive, including for example, malignancy, autoimmune disorders,diabetes, obesity, and infection.

[0007] The protein tyrosine phosphatase (PTP) family of enzymes consistsof more than 100 structurally diverse proteins in vertebrates, includingalmost 40 human PTPs that have in common the conserved 250 amino acidPTP catalytic domain, but which display considerable variation in theirnon-catalytic segments (Charbonneau and Tonks, 1992 Annu. Rev. CellBiol. 8:463-493; Tonks, 1993 Semin. Cell Biol. 4:373-453; Andersen etal., Mol. Cell Biol. 21:7117-36 (2001)). This structural diversitypresumably reflects the diversity of physiological roles of individualPTP family members, which in certain cases have been demonstrated tohave specific functions in growth, development and differentiation(Desai et al., 1996 Cell 84:599-609; Kishihara et al., 1993 Cell74:143-156; Perkins et al., 1992 Cell 70:225-236; Pingel and Thomas,1989 Cell 58:1055-1065; Schultz et al., 1993 Cell 73:1445-1454). The PTPfamily includes receptor-like and non-transmembrane enzymes that exhibitexquisite substrate specificity in vivo and that are involved inregulating a wide variety of cellular signaling pathways (Andersen etal., Mol. Cell. Biol. 21:7117 (2001); Tonks and Neel, Curr. Opin. CellBiol. 13:182 (2001)). PTPs thus participate in a variety of physiologicfunctions, providing a number of opportunities for therapeuticintervention in physiologic processes through alteration (i.e., astatistically significant increase or decrease) or modulation (e.g.,up-regulation or down-regulation) of PTP activity.

[0008] Although recent studies have also generated considerableinformation regarding the structure, expression and regulation of PTPs,the nature of many tyrosine phosphorylated substrates through which thePTPs exert their effects remains to be determined. Studies with alimited number of synthetic phosphopeptide substrates have demonstratedsome differences in the substrate selectivities of different PTPs (Choet al., 1993 Protein Sci. 2: 977-984; Dechert et al., 1995 Eur. J.Biochem. 231:673-681). Analyses of PTP-mediated dephosphorylation of PTPsubstrates suggest that catalytic activity may be favored by thepresence of certain amino acid residues at specific positions in thesubstrate polypeptide relative to the phosphorylated tyrosine residue(Salmeen et al., 2000 Molecular Cell 6:1401; Myers et al., 2001 J. Biol.Chem. 276:47771; Myers et al., 1997 Proc. Natl. Acad. Sci. USA 94:9052;Ruzzene et al., 1993 Eur. J. Biochem. 211:289-295; Zhang et al., 1994Biochemistry 33:2285-2290). Thus, although the physiological relevanceof the substrates used in these studies is unclear, PTPs display acertain level of substrate selectivity in vitro.

[0009] The PTP family of enzymes contains a common evolutionarilyconserved segment of approximately 250 amino acids known as the PTPcatalytic domain. Within this conserved domain is a unique signaturesequence motif, CX₅R (SEQ ID NO:______), that is invariant among allPTPs. In a majority of PTPs, an 11 amino acid conserved sequence([I/V]HCXAGXXR[S/T)G (SEQ ID NO:1)) containing the signature sequencemotif is found. The cysteine residue in this motif is invariant inmembers of the family and is essential for catalysis of thephosphotyrosine dephosphorylation reaction. It functions as anucleophile to attack the phosphate moiety present on a phosphotyrosineresidue of the incoming substrate. If the cysteine residue is altered bysite-directed mutagenesis to serine (e.g., in cysteine-to-serine or “CS”mutants) or alanine (e.g., cysteine-to-alanine or “CA” mutants), theresulting PTP is catalytically deficient but retains the ability tocomplex with, or bind, its substrate, at least in vitro.

[0010] Human T cell protein tyrosine phosphatase (TCPTP) is anontransmembrane PTP. Alternative mRNA splicing results in variation inthe sequence at the extreme C terminus of TCPTP and generates a 45-kDaform (TC45) that is targeted to the nucleus and a 48-kDa variant (TC48)associated with membranes of the endoplasmic reticulum (Hao, et al., J.Biol. Chem. 272:29322-29 (1997). The alternatively spliced forms, TC45and TC48, share the same catalytic domain but differ at their extremecarboxy-termini (Mosinger et al., Proc. Natl. Acad. Sci. USA 89:499-503(1992)). TC45 is characterized by the presence of bipartite nuclearlocalization signal and it is rapidly translocated from the nucleus tothe cytoplasm following EGF stimulation. In the cytoplasm, TC45dephosphorylates the EGF receptor, as well as downstream adaptersincluding p52^(Shc), at the plasma membrane, thereby regulating growthfactor signaling (Tiganis et al., Mol. Cell Biol. 18:1622-34 (1998);Tiganis et al, J. Biol. Chem. 274:27768:75 (1999)). Overexpression ofTC45 has been implicated in inducing p53-dependent, serum starvationindependent, and caspase-mediated apoptosis (Radha et al., FEBS Lett.453:308-12 (1999).

[0011] RNA interference (RNAi) is a polynucleotide sequence-specific,post-transcriptional gene silencing mechanism effected bydouble-stranded RNA that results in degradation of a specific messengerRNA (mRNA), thereby reducing the expression of a desired targetpolypeptide encoded by the mRNA (see, e.g., WO 99/32619; WO 01/75164;U.S. Pat. No. 6,506,559; Fire et al., Nature 391:806-11 (1998); Sharp,Genes Dev. 13:139-41 (1999); Elbashir et al. Nature 411:494-98 (2001);Harborth et al., J. Cell Sci. 114:4557-65 (2001)). RNAi is mediated bydouble-stranded polynucleotides as also described hereinbelow, forexample, double-stranded RNA (dsRNA), having sequences that correspondto exonic sequences encoding portions of the polypeptides for whichexpression is compromised. RNAi reportedly is not effected bydouble-stranded RNA polynucleotides that share sequence identity withintronic or promoter sequences (Elbashir et al., 2001). RNAi pathwayshave been best characterized in Drosophila and Caenorhabditis elegans,but “small interfering RNA” (siRNA) polynucleotides that interfere withexpression of specific polypeptides in higher eukaryotes such as mammals(including humans) have also been considered (e.g., Tuschl, 2001Chembiochem. 2:239-245; Sharp, 2001 Genes Dev. 15:485; Bernstein et al.,2001 RNA 7:1509; Zamore, 2002 Science 296:1265; Plasterk, 2002 Science296:1263; Zamore 2001 Nat. Struct. Biol. 8:746; Matzke et al., 2001Science 293:1080; Scadden et al., 2001 EMBO Rep. 2:1107).

[0012] According to a current non-limiting model, the RNAi pathway isinitiated by ATP-dependent, processive cleavage of long dsRNA intodouble-stranded fragments of about 18-27 (e.g., 19, 20, 21, 22, 23, 24,25, 26, etc.) nucleotide base pairs in length, called small interferingRNAs (siRNAs) (see review by Hutvagner et al., Curr. Opin. Gen. Dev.12:225-32 (2002); Elbashir et al., 2001; Nykänen et al., Cell 107:309-21(2001); Zamore et al., Cell 101:25-33 (2000)). In Drosophila, an enzymeknown as “Dicer” cleaves the longer double-stranded RNA into siRNAs;Dicer belongs to the RNase III family of dsRNA-specific endonucleases(WO 01/68836; Bernstein et al., Nature 409:363-66 (2001)). Furtheraccording to this non-limiting model, the siRNA duplexes areincorporated into a protein complex, followed by ATP-dependent unwindingof the siRNA, which then generates an active RNA-induced silencingcomplex (RISC) (WO 01/68836). The complex recognizes and cleaves atarget RNA that is complementary to the guide strand of the siRNA, thusinterfering with expression of a specific protein (Hutvagner et al.,supra).

[0013] In C. elegans and Drosophila, RNAi may be mediated by longdouble-stranded RNA polynucleotides (WO 99/32619; WO 01/75164; Fire etal., 1998; Clemens et al., Proc. Natl. Acad. Sci. USA 97:6499-6503(2000); Kisielow et al., Biochem. J. 363:1-5 (2002); see also WO01/92513 (RNAi-mediated silencing in yeast)). In mammalian cells,however, transfection with long dsRNA polynucleotides (i.e., greaterthan 30 base pairs) leads to activation of a non-specific sequenceresponse that globally blocks the initiation of protein synthesis andcauses mRNA degradation (Bass, Nature 411:428-29 (2001)). Transfectionof human and other mammalian cells with double-stranded RNAs of about18-27 nucleotide base pairs in length interferes in a sequence-specificmanner with expression of particular polypeptides encoded by messengerRNAs (mRNA) containing corresponding nucleotide sequences (WO 01/75164;Elbashir et al., 2001; Elbashir et al., Genes Dev. 15:188-200 (2001));Harborth et al., J. Cell Sci. 114:4557-65 (2001); Carthew et al., Curr.Opin. Cell Biol. 13:244-48 (2001); Mailand et al., Nature Cell Biol.Advance Online Publication (Mar. 18, 2002); Mailand et al. 2002 NatureCell Biol. 4:317).

[0014] siRNA polynucleotides may offer certain advantages over otherpolynucleotides known to the art for use in sequence-specific alterationor modulation of gene expression to yield altered levels of an encodedpolypeptide product. These advantages include lower effective siRNApolynucleotide concentrations, enhanced siRNA polynucleotide stability,and shorter siRNA polynucleotide oligonucleotide lengths relative tosuch other polynucleotides (e.g., antisense, ribozyme or triplexpolynucleotides). By way of a brief background, “antisense”polynucleotides bind in a sequence-specific manner to target nucleicacids, such as mRNA or DNA, to prevent transcription of DNA ortranslation of the mRNA (see, e.g., U.S. Pat. No. 5,168,053; U.S. Pat.No. 5,190,931; U.S. Pat. No. 5,135,917; U.S. Pat. No. 5,087,617; seealso, e.g., Clusel et al., 1993 Nucl. Acids Res. 21:3405-11, describing“dumbbell” antisense oligonucleotides). “Ribozyme” polynucleotides canbe targeted to any RNA transcript and are capable of catalyticallycleaving such transcripts, thus impairing translation of mRNA (see,e.g., U.S. Pat. No. 5,272,262; U.S. Pat. No. 5,144,019; and U.S. Pat.Nos. 5,168,053, 5,180,818, 5,116,742 and 5,093,246; U.S. Ser. No.2002/193579). “Triplex” DNA molecules refers to single DNA strands thatbind duplex DNA to form a colinear triplex molecule, thereby preventingtranscription (see, e.g., U.S. Pat. No. 5,176,996, describing methodsfor making synthetic oligonucleotides that bind to target sites onduplex DNA). Such triple-stranded structures are unstable and form onlytransiently under physiological conditions. Because single-strandedpolynucleotides do not readily diffuse into cells and are thereforesusceptible to nuclease digestion, development of single-stranded DNAfor antisense or triplex technologies often requires chemically modifiednucleotides to improve stability and absorption by cells. siRNAs, bycontrast, are readily taken up by intact cells, are effective atinterfering with the expression of specific polypeptides atconcentrations that are several orders of magnitude lower than thoserequired for either antisense or ribozyme polynucleotides, and do notrequire the use of chemically modified nucleotides.

[0015] Importantly, despite a number of attempts to devise selectioncriteria for identifying oligonucleotide sequences that will beeffective in siRNA based on features of the desired target mRNA sequence(e.g., percent GC content, position from the translation start codon, orsequence similarities based on an in silico sequence database search forhomologues of the proposed siRNA) it is presently not possible topredict with any degree of confidence which of myriad possible candidatesiRNA sequences that can be generated as nucleotide sequences thatcorrespond to a desired target mRNA (e.g., dsRNA of about 18-27nucleotide base pairs) will in fact exhibit siRNA activity (i.e.,interference with expression of the polypeptide encoded by the mRNA).Instead, individual specific candidate siRNA polynucleotide oroligonucleotide sequences must be generated and tested to determinewhether interference with expression of a desired polypeptide target canbe effected. Accordingly, no routine method exists in the art fordesigning a siRNA polynucleotide that is, with certainty, capable ofspecifically altering the expression of a given PTP polypeptide, andthus for the overwhelming majority of PTPs no effective siRNApolynucleotide sequences are presently known.

[0016] Currently, therefore, desirable goals for therapeutic regulationof biological signal transduction include modulation of PTP (e.g.,TCPTP, PTP1B, DSP-3, or other PTP)-mediated cellular events include,inter alia, inhibition or potentiation of interactions among PTP-bindingmolecules, substrates and binding partners, or of other agents thatregulate PTP activities. Accordingly, a need exists in the art for animproved ability to intervene in the regulation of phosphotyrosinesignaling, including regulating by altering PTP catalytic activity, PTPbinding to substrate molecules, and/or -encoding gene expression. Anincreased ability to so regulate may facilitate the development ofmethods for modulating the activity of proteins involved inphosphotyrosine signaling pathways and for treating conditionsassociated with such pathways. The present invention fulfills theseneeds and further provides other related advantages.

SUMMARY OF THE INVENTION

[0017] The present invention relates to compositions and methods,including specific siRNA polynucleotides comprising nucleotide sequencesdisclosed herein, for modulating TCPTP. It is therefore an aspect of theinvention to provide an isolated small interfering RNA (siRNA)polynucleotide, comprising at least one nucleotide sequence selectedfrom SEQ ID NOS:28-31, 33-36, 38-41, and 68-71, which in certain furtherembodiments comprises at least one nucleotide sequence selected from thegroup consisting of SEQ ID NOS: 28-31, 33-36, 38-41, and 68-71 and thecomplementary polynucleotide thereto, and which siRNA polynucleotide incertain further embodiments is capable of interfering with expression ofa TCPTP polypeptide, wherein the TCPTP polypeptide comprises an aminoacid sequence as set forth in a sequence selected from GenBank Acc. Nos.M25393, NM_(—)002828, NM_(—)080422, and SEQ ID NOS:4, 12, and 14. Inanother embodiment the siRNA polynucleotide differs by one, two, threeor four nucleotides at any of positions 1-19 of a sequence selected fromthe sequences set forth in SEQ ID NOS: 28-31, 33-36, 38-41, and 68-71.In another embodiment the siRNA polynucleotide differs by at least two,three or four nucleotides at any of positions 1-19 of a sequenceselected from the sequences set forth in SEQ ID NOS: 28-31, 33-36,38-41, and 68-71. The invention provides in other embodiments anisolated siRNA polynucleotide comprising a nucleotide sequence accordingto SEQ ID NO: 28, or the complement thereof; SEQ ID NO: 33, or thecomplement thereof; SEQ ID NO: 38, or the complement thereof; and SEQ IDNO: 68, or the complement thereof. In certain embodiments the siRNApolynucleotide comprises at least one synthetic nucleotide analogue of anaturally occurring nucleotide. In another embodiment the siRNApolynucleotide is linked to a detectable label, which in certain furtherembodiments is a reporter molecule that in certain further embodimentsis a dye, a radionuclide, a luminescent group, a fluorescent group, orbiotin. In a further embodiment the fluorescent group is fluoresceinisothiocyanate, and in a distinct further embodiment the detectablelabel is a magnetic particle. The invention also provides apharmaceutical composition comprising any of the above described siRNApolynucleotides and a physiologically acceptable carrier, which carrierin a further embodiment comprises a liposome.

[0018] It is another aspect of the invention to provide a recombinantnucleic acid construct comprising a polynucleotide that is capable ofdirecting transcription of a small interfering RNA (siRNA), thepolynucleotide comprising (i) a first promoter; (ii) a second promoter;and (iii) at least one DNA polynucleotide segment comprising at leastone nucleotide sequence selected from SEQ ID NOS: 28-31, 33-36, 38-41,and 68-71, or a complement thereto, wherein each DNA polynucleotidesegment and its complement are operably linked to at least one of thefirst and second promoters, and wherein the promoters are oriented todirect transcription of the DNA polynucleotide segment and its reversecomplement. In another embodiment the construct comprises at least oneenhancer that is selected from a first enhancer operably linked to thefirst promoter and a second enhancer operably linked to the secondpromoter. In another embodiment the construct comprises at least onetranscriptional terminator that is selected from (i) a firsttranscriptional terminator that is positioned in the construct toterminate transcription directed by the first promoter and (ii) a secondtranscriptional terminator that is positioned in the construct toterminate transcription directed by the second promoter. In anotherembodiment the siRNA is capable of interfering with expression of aTCPTP polypeptide, wherein the TCPTP polypeptide comprises an amino acidsequence as set forth in a sequence selected from GenBank Acc. Nos.M25393, NM_(—)002828, and NM_(—)080422, SEQ ID NOS:4, 12, and 14.

[0019] According to another embodiment of the invention there isprovided a recombinant nucleic acid construct comprising apolynucleotide that is capable of directing transcription of a smallinterfering RNA (siRNA), the polynucleotide comprising at least onepromoter and a DNA polynucleotide segment, wherein the DNApolynucleotide segment is operably linked to the promoter, and whereinthe DNA polynucleotide segment comprises (i) at least one DNApolynucleotide that comprises at least one nucleotide sequence selectedfrom SEQ ID NOS: 28-31, 33-36, 38-41, and 68-71, or a complementthereto; (ii) a spacer sequence comprising at least 4 nucleotidesoperably linked to the DNA polynucleotide of (i); and (iii) the reversecomplement of the DNA polynucleotide of (i) operably linked to thespacer sequence. In a further embodiment the siRNA comprises an overhangof at least one and no more than four nucleotides, the overhang beinglocated immediately 3′ to (iii). In another embodiment the spacersequence comprises at least 9 nucleotides, and in another embodiment thespacer sequence comprises two uridine nucleotides that are contiguouswith (iii). In another embodiment the recombinant nucleic acid constructcomprises at least one transcriptional terminator that is operablylinked to the DNA polynucleotide segment. In another related embodimentthe invention provides a host cell transformed or transfected with therecombinant nucleic acid constructs just described.

[0020] Turning to another embodiment, there is provided by the presentinvention a pharmaceutical composition comprising an siRNApolynucleotide and a physiologically acceptable carrier, wherein thesiRNA polynucleotide is selected from (i) an RNA polynucleotide whichcomprises at least one nucleotide sequence selected from the groupconsisting of SEQ ID NOS: 28-31, 33-36, 38-41, and 68-71, (ii) an RNApolynucleotide that comprises at least one nucleotide sequence selectedfrom SEQ ID NOS: 28-31, 33-36, 38-41, and 68-71and the complementarypolynucleotide thereto, (iii) an RNA polynucleotide according to (i) or(ii) wherein the nucleotide sequence of the siRNA polynucleotide differsby one, two or three nucleotides at any of positions 1-19 of a sequenceselected from the sequences set forth in SEQ ID NOS: 28-31, 33-36,38-41, and 68-71, and (iv) an RNA polynucleotide according to (i) or(ii) wherein the nucleotide sequence of the siRNA polynucleotide differsby two, three or four nucleotides at any of positions 1-19 of a sequenceselected from the sequences set forth in SEQ ID NOS: 28-31, 33-36,38-41, and 68-71. In another related embodiment the carrier comprises aliposome.

[0021] According to another aspect of the invention, there is provided amethod for interfering with expression of a TCPTP polypeptide, orvariant thereof, comprising contacting a subject that comprises at leastone cell which is capable of expressing a TCPTP polypeptide with a siRNApolynucleotide for a time and under conditions sufficient to interferewith TCPTP polypeptide expression, wherein (a) the PTP1B polypeptidecomprises an amino acid sequence as set forth in a sequence selectedfrom GenBank Acc. Nos. M25393, NM_(—)002828, and NM_(—)080422, SEQ IDNOS:4, 12, and 14, (b) the siRNA polynucleotide is selected from thegroup consisting of (i) an RNA polynucleotide which comprises at leastone nucleotide sequence selected from the group consisting of SEQ IDNOS: 28-31, 33-36, 38-41, and 68-71, (ii) an RNA polynucleotide thatcomprises at least one nucleotide sequence selected from the groupconsisting of SEQ ID NOS: 28-31, 33-36, 38-41, and 68-71and thecomplementary polynucleotide thereto, (iii) an RNA polynucleotideaccording to (i) or (ii) wherein the nucleotide sequence of the siRNApolynucleotide differs by one, two or three nucleotides at any ofpositions 1-19 of a sequence selected from the group consisting of thesequences set forth in SEQ ID NOS: 28-31, 33-36, 38-41, and 68-71, and(iv) an RNA polynucleotide according to (i) or (ii) wherein thenucleotide sequence of the siRNA polynucleotide differs by two, three orfour nucleotides at any of positions 1-19 of a sequence selected fromthe group consisting of the sequences set forth in SEQ ID NOS: 28-31,33-36, 38-41, and 68-71.

[0022] In another embodiment there is provided a method for interferingwith expression of a TCPTP polypeptide that comprises an amino acidsequence as set forth in a sequence selected from the group consistingof GenBank Acc. Nos. M25393, NM_(—)002828, and NM_(—)080422, SEQ IDNOS:4, 12, and 14, or a variant of said TCPTP polypeptide, said methodcomprising contacting, under conditions and for a time sufficient tointerfere with TCPTP polypeptide expression, (i) a subject thatcomprises at least one cell that is capable of expressing the TCPTPpolypeptide, and (ii) a recombinant nucleic acid construct according tothe above described aspects and embodiments.

[0023] The invention also provides in certain embodiments a method foridentifying a component of a TCPTP signal transduction pathwaycomprising A. contacting a siRNA polynucleotide and a first biologicalsample comprising at least one cell that is capable of expressing aTCPTP polypeptide, or a variant of said TCPTP polypeptide, underconditions and for a time sufficient for TCPTP expression when the siRNApolynucleotide is not present, wherein (1) the TCPTP polypeptidecomprises an amino acid sequence as set forth in a sequence selectedfrom GenBank Acc. Nos. M25393, NM_(—)002828, and NM_(—)080422, SEQ IDNOS:4, 12, and 14, (2) the siRNA polynucleotide is selected from (i) anRNA polynucleotide which comprises at least one nucleotide sequenceselected from the group consisting of SEQ ID NOS: 28-31, 33-36, 38-41,and 68-71, (ii) an RNA polynucleotide that comprises at least onenucleotide sequence selected from the group consisting of SEQ ID NOS:28-31, 33-36, 38-41, and 68-71and the complementary polynucleotidethereto, (iii) an RNA polynucleotide according to (i) or (ii) whereinthe nucleotide sequence of the siRNA polynucleotide differs by one, twoor three nucleotides at any of positions 1-19 of a sequence selectedfrom the sequences set forth in SEQ ID NOS: 28-31, 33-36, 38-41, and68-71, and (iv) an RNA polynucleotide according to (i) or (ii) whereinthe nucleotide sequence of the siRNA polynucleotide differs by two,three or four nucleotides at any of positions 1-19 of a sequenceselected from the sequences set forth in SEQ ID NOS: 28-31, 33-36,38-41, and 68-71; and B. comparing a level of phosphorylation of atleast one protein that is capable of being phosphorylated in the cellwith a level of phosphorylation of the protein in a control sample thathas not been contacted with the siRNA polynucleotide, wherein an alteredlevel of phosphorylation of the protein in the presence of the siRNApolynucleotide relative to the level of phosphorylation of the proteinin an absence of the siRNA polynucleotide indicates that the protein isa component of the TCPTP signal transduction pathway. In certainembodiments the signal transduction pathway comprises a Jak2 kinase.

[0024] Another aspect of the invention provides a method for modulatingan insulin receptor protein phosphorylation state in a cell, comprisingcontacting the cell with a siRNA polynucleotide under conditions and fora time sufficient to interfere with expression of a TCPTP polypeptide,wherein (a) the TCPTP polypeptide comprises an amino acid sequence asset forth in a sequence selected from the group consisting of GenBankAcc. Nos. M25393, NM_(—)002828, and NM_(—)080422, SEQ ID NOS:4, 12, and14, (b) the siRNA polynucleotide is selected from the group consistingof (i) an RNA polynucleotide which comprises at least one nucleotidesequence selected from the group consisting of SEQ ID NOS: 28-31, 33-36,38-41, and 68-71, or the complements thereof, (ii) an RNA polynucleotidethat comprises at least one nucleotide sequence selected from the groupconsisting of SEQ ID NOS: 28-31, 33-36, 38-41, and 68-71and thecomplementary polynucleotide thereto, (iii) an RNA polynucleotideaccording to (i) or (ii) wherein the nucleotide sequence of the siRNApolynucleotide differs by one, two or three nucleotides at any ofpositions 1-19 of a sequence selected from the group consisting of thesequences set forth in SEQ ID NOS: 28-31, 33-36, 38-41, and 68-71, and(iv) an RNA polynucleotide according to (i) or (ii) wherein thenucleotide sequence of the siRNA polynucleotide differs by two, three orfour nucleotides at any of positions 1-19 of a sequence selected fromthe group consisting of the sequences set forth in SEQ ID NOS: 28-31,33-36, 38-41, and 68-71; and (c) the insulin receptor protein comprisesa polypeptide which comprises an amino acid sequence selected from thegroup consisting of SEQ ID NOS:______-______, or a variant thereof.

[0025] Another aspect of the invention pertains to a method for alteringJak2 protein phosphorylation state in a cell, comprising contacting thecell with a siRNA polynucleotide under conditions and for a timesufficient to interfere with expression of a TCPTP polypeptide, wherein(a) the TCPTP polypeptide comprises an amino acid sequence as set forthin a sequence selected from GenBank Acc. Nos. M25393, NM_(—)002828, andNM_(—)080422, SEQ ID NOS:4, 12, and 14, (b) the siRNA polynucleotide isselected from (i) an RNA polynucleotide which comprises at least onenucleotide sequence selected from SEQ ID NOS: 28-31, 33-36, 38-41, and68-71, or the complements thereof, (ii) an RNA polynucleotide thatcomprises at least one nucleotide sequence selected from SEQ ID NOS:28-31, 33-36, 38-41, and 68-71 and the complementary polynucleotidethereto, (iii) an RNA polynucleotide according to (i) or (ii) whereinthe nucleotide sequence of the siRNA polynucleotide differs by one, twoor three nucleotides at any of positions 1-19 of a sequence selectedfrom the sequences set forth in SEQ ID NOS: 28-31, 33-36, 38-41, and68-71, and (iv) an RNA polynucleotide according to (i) or (ii) whereinthe nucleotide sequence of the siRNA polynucleotide differs by two,three or four nucleotides at any of positions 1-19 of a sequenceselected from the sequences set forth in SEQ ID NOS: 28-31, 33-36,38-41, and 68-71; and (c) the Jak2 protein comprises a polypeptide whichcomprises an amino acid sequence selected from the group consisting ofSEQ ID NOS:______-______, or a variant thereof. In another embodimentthere is provided a method for treating a Jak2-associated disordercomprising administering to a subject in need thereof a pharmaceuticalcomposition as described above, wherein the siRNA polynucleotideinhibits expression of a TCPTP polypeptide, or a variant thereof. In afurther embodiment the Jak2-associated disorder is selected from thegroup consisting of diabetes, obesity, hyperglycemia-induced apoptosis,inflammation, and a neurodegenerative disorder. In another aspect theinvention provides a small interfering RNA (siRNA) polynucleotide,comprising in certain embodiments at least one nucleotide sequenceselected from SEQ ID NOS:28-31, 33-36, 38-41, and 68-71. Certain furtherembodiments relate to isolated siRNA polynucleotides that comprisenucleotide sequences having the above recited SEQ ID NOS, includingcompositions and methods for producing and therapeutically using suchsiRNA.

[0026] These and other embodiments of the present invention will becomeapparent upon reference to the following detailed description andattached drawings. All references disclosed herein are herebyincorporated by reference in their entireties as if each wasincorporated individually. Also incorporated by reference are co-pendingapplications, Ser. No. ______ and Ser. No. ______ (attorney docketnumbers 200125.441 and 200125.449, respectively), which have been filedconcurrently.

BRIEF DESCRIPTION OF THE DRAWINGS

[0027]FIG. 1 illustrates insulin-induced activation of PKB/Akt in HepG2cells following ablation of TC45 by RNA interference. FIG. 1A representsan immunoblot of serum-deprived Rat-1 and HEPG2 cells that were exposedto varying concentrations of insulin (INS) as shown. The insulinreceptor (IR) was immunoprecipitated from cell lysates with an anti-IR-βantibody followed by immunoblotting with an anti-phosphotyrosineantibody (pY) (top panel); an anti-pYpY^(1162/1163)-IR-β antibody(middle panel); and an anti-IR β antibody. FIG. 1B represents animmunoblot of HepG2 cell lysates prepared from cells that wereuntransfected (control) or transfected with TCPTP1 siRNA (SEQ IDNO:______) (+siRNA). The lysates were immunoblotted with ananti-phospho-PKB/Akt antibody (p-AKT) (first immunoblot); anti-PKB/Aktantibody (AKT) (second immunoblot); anti-TC45 (TC45) antibody (thirdimmunoblot); and an anti-PTP1B antibody (PTP1B). FIG. 1C represents adensitometric analysis of the gel image to illustrate the ratio ofphosphorylated PKB/Akt to total PKB/Akt.

[0028]FIG. 2 provides an immunoblot indicating that tyrosinephosphorylated IR-β is a substrate of TC45. HepG2 cells overexpressingwild-type (WT) or substrate trapping mutant (DA) forms of PTP1B (1B) andTC45 were either not treated with insulin (−INS) or stimulated withinsulin for 5 minutes (+INS), lysed, separated by SDS-PAGE, andimmunoprecipitated with anti-PTP1B antibody (FG6) or anti-TC45 antibody(CF4). The immunoprecipitates were immunoblotted with an anti-IR-βantibody (top panel, FIG. 2A); anti-PTP1B antibody FG6 (middle panel,FIG. 2A); and anti-TCPTP antibody CF4 (bottom panel, FIG. 2A). FIG. 2Bdepicts immunoblots of HepG2 cells that were serum-starved anduntransfected (control) or transfected with TC45 siRNA (100 nM) and thenstimulated with 10 nM insulin (INS) for the indicated times. The insulinreceptor was immunoprecipitated from cell lysates with an anti-IR-βantibody, which was then immunoblotted with the following antibodies:anti-phosphotyrosine (p-Tyr) (first immunoblot); anti-pY⁹⁷²-IR-β (secondimmunoblot); anti-pYpY^(1162/1163)-IR-β (third immunoblot); andanti-IR-β (fourth immunoblot). FIG. 2C presents densitometric analysesof the gel image to show the ratio of phosphorylated IR-β, to total IR-βfor total phosphotyrosine (top panel); phosphorylation of Tyr 972(middle panel); and phosphorylation of the activation loop tyrosines1162 and 1163 (lower panel).

[0029]FIG. 3 depicts the results of an ELISA in which the level ofinsulin receptor (IR) phosphorylated tyrosine was measured in 293-HEKHIR cells transfected with 0, 0.5, 3, or 10 nM hTCPTP1.4 siRNA (TC1.4,SEQ ID NO:______) (FIG. 3A) and mPTP1B1.1b siRNA (M1.1, SEQ IDNO:______) (FIG. 3B). Seventy-two hours after transfection, cells wereexposed to insulin for 7 minutes at the designated concentrations. Celllysates were prepared and coated onto 96-well plates and probed with ananti-pY-IR-β antibody.

DETAILED DESCRIPTION OF THE INVENTION

[0030] The present invention is directed in part to the unexpecteddiscovery of short RNA polynucleotide sequences that are capable ofspecifically modulating expression of a desired TC-PTP polypeptide, suchas a human or murine TC-PTP polypeptide (e.g., GenBank Accession Nos.M25393 (SEQ ID NOS: ______-______); M81478 (SEQ ID NO: ______); M80737(SEQ ID NO: ______); M81477 (SEQ ID NOS: ______-______); X58828 (SEQ IDNOS: ______-______); NM_(—)002828; and TC45 (e.g. NM_(—)080422 (SEQ IDNOS: ______ and ______); Andersen et al., 2001 Mol. Cell. Biol.21:7117), or variant thereof. Without wishing to be bound by theory, theRNA polynucleotides of the present invention specifically reduceexpression of a desired target polypeptide through recruitment of smallinterfering RNA (siRNA) mechanisms. In particular, and as described ingreater detail herein, according to the present invention there areprovided compositions and methods that relate to the surprisingidentification of certain specific RNAi oligonucleotide sequences of 19,20, 21, 22, 23, 24, 25, 26 or 27 nucleotides that can be derived fromcorresponding polynucleotide sequences encoding the desired TCPTP targetpolypeptide. These sequences cannot be predicted through any algorithm,sequence alignment routine, or other systematic paradigm, but mustinstead be obtained through generation and functional testing for RNAiactivity of actual candidate oligonucleotides, such as those disclosedfor the first time herein.

[0031] In preferred embodiments of the invention, the siRNApolynucleotide interferes with expression of a TCPTP target polypeptideor a variant thereof, and comprises a RNA oligonucleotide or RNApolynucleotide uniquely corresponding in its nucleotide base sequence tothe sequence of a portion of a target polynucleotide encoding the targetpolypeptide, for instance, a target mRNA sequence or an exonic sequenceencoding such mRNA. The invention relates in preferred embodiments tosiRNA polynucleotides that interfere with expression of specificpolypeptides in mammals, which in certain particularly preferredembodiments are humans and in certain other particularly preferredembodiments are non-human mammals. Hence, according to non-limitingtheory, the siRNA polynucleotides of the present invention directsequence-specific degradation of mRNA encoding a desired TCPTP.

[0032] PTP associated disorders include, for example, diabetes mellitus,obesity, impaired glucose tolerance and other metabolic disorderswherein alteration of a TCPTP or of a TCPTP signaling pathway componentis associated with such a disorder. The protein tyrosine phosphataseTCPTP exists in two alternatively spliced forms, TC45 and TC48, thatshare the same catalytic domain but differ at their extremecarboxy-termini (Mosinger et al., Proc. Natl. Acad. Sci. USA 89:499-503(1992)). Insulin-induced oxidation and inactivation of TC45 suggeststhat it may function as a negative regulator of insulin signaling (seeU.S. Ser. No. 10/366,547; see also, Galic et al., Mol. Cell Biol.23:2096-108 (2003)). The effect of siRNA interference with expression ofa component in the signal transduction pathway induced by insulin, forexample, may be evaluated by determining the level of tyrosinephosphorylation of insulin receptor beta (IR-β) and/or of the downstreamsignaling molecule PKB/Akt and/or of any other downstream polypeptidethat may be a component of a particular signal transduction pathway asprovided herein. As used herein, TCPTP is understood to include TCPTPand its alternatively spliced forms.

SiRNA Polynucleotides

[0033] As used herein, the term “siRNA” means either: (i) a doublestranded RNA oligonucleotide, or polynucleotide, that is 18 base pairs,19 base pairs, 20 base pairs, 21 base pairs, 22 base pairs, 23 basepairs, 24 base pairs, 25 base pairs, 26 base pairs, 27 base pairs, 28base pairs, 29 base pairs or 30 base pairs in length and that is capableof interfering with expression and activity of a TCPTP polypeptide, or avariant of the TCPTP polypeptide, wherein a single strand of the siRNAcomprises a portion of a RNA polynucleotide sequence that encodes theTCPTP polypeptide, its variant, or a complementary sequence thereto;(ii) a single stranded oligonucleotide, or polynucleotide of 18nucleotides, 19 nucleotides, 20 nucleotides, 21 nucleotides, 22nucleotides, 23 nucleotides, 24 nucleotides, 25 nucleotides, 26nucleotides, 27 nucleotides, 28 nucleotides, 29 nucleotides or 30nucleotides in length and that is either capable of interfering withexpression and/or activity of a target TCPTP polypeptide, or a variantof the TCPTP polypeptide, or that anneals to a complementary sequence toresult in a dsRNA that is capable of interfering with target polypeptideexpression, wherein such single stranded oligonucleotide comprises aportion of a RNA polynucleotide sequence that encodes the TCPTPpolypeptide, its variant, or a complementary sequence thereto; or (iii)an oligonucleotide, or polynucleotide, of either (i) or (ii) abovewherein such oligonucleotide, or polynucleotide, has one, two, three orfour nucleic acid alterations or substitutions therein.

[0034] A siRNA polynucleotide is a RNA nucleic acid molecule thatmediates the effect of RNA interference, a post-transcriptional genesilencing mechanism. A siRNA polynucleotide preferably comprises adouble-stranded RNA (dsRNA) but is not intended to be so limited and maycomprise a single-stranded RNA (see, e.g., Martinez et al. Cell110:563-74 (2002)). A siRNA polynucleotide may comprise other naturallyoccurring, recombinant, or synthetic single-stranded or double-strandedpolymers of nucleotides (ribonucleotides or deoxyribonucleotides or acombination of both) and/or nucleotide analogues as provided herein(e.g., an oligonucleotide or polynucleotide or the like, typically in 5′to 3′ phosphodiester linkage). Accordingly it will be appreciated thatcertain exemplary sequences disclosed herein as DNA sequences capable ofdirecting the transcription of the subject invention siRNApolynucleotides are also intended to describe the corresponding RNAsequences and their complements, given the well established principlesof complementary nucleotide base-pairing. A siRNA may be transcribedusing as a template a DNA (genomic, cDNA, or synthetic) that contains aRNA polymerase promoter, for example, a U6 promoter or the H1 RNApolymerase III promoter, or the siRNA may be a synthetically derived RNAmolecule. In certain embodiments the subject invention siRNApolynucleotide may have blunt ends, that is, each nucleotide in onestrand of the duplex is perfectly complementary (e.g., by Watson-Crickbase-pairing) with a nucleotide of the opposite strand. In certain otherembodiments, at least one strand of the subject invention siRNApolynucleotide has at least one, and preferably two nucleotides that“overhang” (i.e., that do not base pair with a complementary base in theopposing strand) at the 3′ end of either strand, or preferably bothstrands, of the siRNA polynucleotide. In a preferred embodiment of theinvention, each strand of the siRNA polynucleotide duplex has atwo-nucleotide overhang at the 3′ end. The two-nucleotide overhang ispreferably a thymidine dinucleotide (TT) but may also comprise otherbases, for example, a TC dinucleotide or a TG dinucleotide, or any otherdinucleotide. For a discussion of 3′ ends of siRNA polynucleotides see,e.g., WO 01/75164.

[0035] Preferred siRNA polynucleotides comprise double-strandedoligomeric nucleotides of about 18-30 nucleotide base pairs, preferablyabout 18, 19, 20, 21, 22, 23, 24, 25, 26, or 27 base pairs, and in otherpreferred embodiments about 19, 20, 21, 22 or 23 base pairs, or about 27base pairs, whereby the use of “about” indicates, as described above,that in certain embodiments and under certain conditions the processivecleavage steps that may give rise to functional siRNA polynucleotidesthat are capable of interfering with expression of a selectedpolypeptide may not be absolutely efficient. Hence, siRNApolynucleotides, for instance, of “about” 18, 19, 20, 21, 22, 23, 24, or25 base pairs may include one or more siRNA polynucleotide moleculesthat may differ (e.g., by nucleotide insertion or deletion) in length byone, two, three or four base pairs, by way of non-limiting theory as aconsequence of variability in processing, in biosynthesis, or inartificial synthesis. The contemplated siRNA polynucleotides of thepresent invention may also comprise a polynucleotide sequence thatexhibits variability by differing (e.g., by nucleotide substitution,including transition or transversion) at one, two, three or fournucleotides from a particular sequence, the differences occurring at anyof positions 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17,18, or 19 of a particular siRNA polynucleotide sequence, or at positions20, 21, 22, 23, 24, 25, 26, or 27 of siRNA polynucleotides depending onthe length of the molecule, whether situated in a sense or in anantisense strand of the double-stranded polynucleotide. The nucleotidesubstitution may be found only in one strand, by way of example in theantisense strand, of a double-stranded polynucleotide, and thecomplementary nucleotide with which the substitute nucleotide wouldtypically form hydrogen bond base pairing may not necessarily becorrespondingly substituted in the sense strand. In preferredembodiments, the siRNA polynucleotides are homogeneous with respect to aspecific nucleotide sequence. As described herein, preferred siRNApolynucleotides interfere with expression of a TCPTP polypeptide. Thesepolynucleotides may also find uses as probes or primers.

[0036] Polynucleotides that are siRNA polynucleotides of the presentinvention may in certain embodiments be derived from a single-strandedpolynucleotide that comprises a single-stranded oligonucleotide fragment(e.g., of about 18-30 nucleotides, which should be understood to includeany whole integer of nucleotides including and between 18 and 30) andits reverse complement, typically separated by a spacer sequence.According to certain such embodiments, cleavage of the spacer providesthe single-stranded oligonucleotide fragment and its reverse complement,such that they may anneal to form (optionally with additional processingsteps that may result in addition or removal of one, two, three or morenucleotides from the 3′ end and/or the 5′ end of either or both strands)the double-stranded siRNA polynucleotide of the present invention. Incertain embodiments the spacer is of a length that permits the fragmentand its reverse complement to anneal and form a double-strandedstructure (e.g., like a hairpin polynucleotide) prior to cleavage of thespacer (and, optionally, subsequent processing steps that may result inaddition or removal of one, two, three, four, or more nucleotides fromthe 3′ end and/or the 5′ end of either or both strands). A spacersequence may therefore be any polynucleotide sequence as provided hereinthat is situated between two complementary polynucleotide sequenceregions which, when annealed into a double-stranded nucleic acid,comprise a siRNA polynucleotide. Preferably a spacer sequence comprisesat least 4 nucleotides, although in certain embodiments the spacer maycomprise 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16,17, 18, 19, 20,21-25, 26-30, 31-40, 41-50, 51-70, 71-90, 91-110, 111-150, 151-200 ormore nucleotides. Examples of siRNA polynucleotides derived from asingle nucleotide strand comprising two complementary nucleotidesequences separated by a spacer have been described (e.g., Brummelkampet al., 2002 Science 296:550; Paddison et al., 2002 Genes Develop.16:948; Paul et al. Nat. Biotechnol. 20:505-508 (2002); Grabarek et al.,BioTechniques 34:734-44 (2003)).

[0037] Polynucleotide variants may contain one or more substitutions,additions, deletions, and/or insertions such that the activity of thesiRNA polynucleotide is not substantially diminished, as describedabove. The effect on the activity of the siRNA polynucleotide maygenerally be assessed as described herein or using conventional methods.Variants preferably exhibit at least about 75%, 78%, 80%, 85%, 87%, 88%or 89% identity and more preferably at least about 90%, 92%, 95%, 96%,97%, 98%, or 99% identity to a portion of a polynucleotide sequence thatencodes a native TCPTP. The percent identity may be readily determinedby comparing sequences of the polynucleotides to the correspondingportion of the TCPTP polynucleotide, using any method including usingcomputer algorithms well known to those having ordinary skill in theart, such as Align or the BLAST algorithm (Altschul, J. Mol. Biol.219:555-565, 1991; Henikoff and Henikoff, Proc. Natl. Acad. Sci. USA89:10915-10919, 1992), which is available at the NCBI website (see[online] Internet:<URL: http://www/ncbi.nlm.nih.gov/cgi-bin/BLAST).Default parameters may be used.

[0038] Certain siRNA polynucleotide variants are substantiallyhomologous to a portion of a native TCPTP gene. Single-stranded nucleicacids derived (e.g., by thermal denaturation) from such polynucleotidevariants are capable of hybridizing under moderately stringentconditions to a naturally occurring DNA or RNA sequence encoding anative TCPTP polypeptide (or a complementary sequence). A polynucleotidethat detectably hybridizes under moderately stringent conditions mayhave a nucleotide sequence that includes at least 10 consecutivenucleotides, more preferably 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21,22, 23, 24, 25, 26, 27, 28, 29 or 30 consecutive nucleotidescomplementary to a particular polynucleotide. In certain preferredembodiments such a sequence (or its complement) will be unique to aTCPTP polypeptide for which interference with expression is desired, andin certain other embodiments the sequence (or its complement) may beshared by TCPTP and one or more PTPs for which interference withpolypeptide expression is desired.

[0039] Suitable moderately stringent conditions include, for example,pre-washing in a solution of 5×SSC, 0.5% SDS, 1.0 mM EDTA (pH 8.0);hybridizing at 50° C.-70° C., 5×SSC for 1-16 hours (e.g., overnight);followed by washing once or twice at 22-65° C. for 20-40 minutes withone or more each of 2×, 0.5× and 0.2×SSC containing 0.05-0.1% SDS. Foradditional stringency, conditions may include a wash in 0.1×SSC and 0.1%SDS at 50-60° C. for 15-40 minutes. As known to those having ordinaryskill in the art, variations in stringency of hybridization conditionsmay be achieved by altering the time, temperature, and/or concentrationof the solutions used for pre-hybridization, hybridization, and washsteps. Suitable conditions may also depend in part on the particularnucleotide sequences of the probe used, and of the blotted, probandnucleic acid sample. Accordingly, it will be appreciated that suitablystringent conditions can be readily selected without undueexperimentation when a desired selectivity of the probe is identified,based on its ability to hybridize to one or more certain probandsequences while not hybridizing to certain other proband sequences.

[0040] Sequence specific siRNA polynucleotides of the present inventionmay be designed using one or more of several criteria. For example, todesign a siRNA polynucleotide that has 19 consecutive nucleotidesidentical to a sequence encoding a polypeptide of interest (e.g., TCPTPand other polypeptides described herein), the open reading frame of thepolynucleotide sequence may be scanned for 21-base sequences that haveone or more of the following characteristics: (1) an A+T/G+C ratio ofapproximately 1:1 but no greater than 2:1 or 1:2; (2) an AA dinucleotideor a CA dinucleotide at the 5′ end; (3) an internal hairpin loop meltingtemperature less than 55° C.; (4) a homodimer melting temperature ofless than 37° C. (melting temperature calculations as described in (3)and (4) can be determined using computer software known to those skilledin the art); (5) a sequence of at least 16 consecutive nucleotides notidentified as being present in any other known polynucleotide sequence(such an evaluation can be readily determined using computer programsavailable to a skilled artisan such as BLAST to search publiclyavailable databases). Alternatively, an siRNA polynucleotide sequencemay be designed and chosen using a computer software availablecommercially from various vendors (e.g., OligoEngine™ (Seattle, Wash.);Dharmacon, Inc. (Lafayette, Colo.); Ambion Inc. (Austin, Tex.); andQIAGEN, Inc. (Valencia, Calif.)). (See also Elbashir et al., Genes &Development 15:188-200 (2000); Elbashir et al., Nature 411:494-98(2001); and [online]Internet:URL<http://www.mpibpc.gwdg.de/abteilungen/100/105/Tuschl_MIV2(3)_(—)2002.pdf.)The siRNA polynucleotides may then be tested for their ability tointerfere with the expression of the target polypeptide according tomethods known in the art and described herein. The determination of theeffectiveness of an siRNA polynucleotide includes not only considerationof its ability to interfere with polypeptide expression but alsoincludes consideration of whether the siRNA polynucleotide manifestsundesirably toxic effects, for example, apoptosis of a cell for whichcell death is not a desired effect of RNA interference.

[0041] It should be appreciated that not all siRNAs designed using theabove methods will be effective at silencing or interfering withexpression of a TCPTP target polypeptide. And further, that the siRNAswill effect silencing to different degrees. Such siRNAs must be testedfor their effectiveness, and selections made therefrom based on theability of a given siRNA to interfere with or modulate (e.g., decreasein a statistically significant manner) the expression of the target.Accordingly, identification of specific siRNA polynucleotide sequencesthat are capable of interfering with expression of a TCPTP polypeptiderequires production and testing of each siRNA, as demonstrated ingreater detail below (see Examples).

[0042] Furthermore, not all siRNAs that interfere with proteinexpression will have a physiologically important effect. The inventorshere have designed, and describe herein, physiologically relevant assaysfor measuring the influence of modulated target polypeptide expression,for instance, cellular proliferation, induction of apoptosis, and/oraltered levels of protein tyrosine phosphorylation (e.g., insulinreceptor phosphorylation), to determine if the levels of interferencewith target protein expression that were observed using the siRNAs ofthe invention have clinically relevant significance. Additionally, andaccording to non-limiting theory, the invention contemplates altered(e.g., decreased or increased in a statistically significant manner)expression levels of one or more polypeptides of interest, and/oraltered (i.e., increased or decreased) phosphorylation levels of one ormore phosphoproteins of interest, which altered levels may result fromimpairment of TCPTP polypeptide expression and/or cellular compensatorymechanisms that are induced in response to RNAi-mediated inhibition of aspecific target polypeptide expression.

[0043] Persons having ordinary skill in the art will also readilyappreciate that as a result of the degeneracy of the genetic code, manynucleotide sequences may encode a polypeptide as described herein. Thatis, an amino acid may be encoded by one of several different codons anda person skilled in the art can readily determine that while oneparticular nucleotide sequence may differ from another (which may bedetermined by alignment methods disclosed herein and known in the art),the sequences may encode polypeptides with identical amino acidsequences. By way of example, the amino acid leucine in a polypeptidemay be encoded by one of six different codons (TTA, TTG, CTT, CTC, CTA,and CTG) as can serine (TCT, TCC, TCA, TCG, AGT, and AGC). Other aminoacids, such as proline, alanine, and valine, for example, may be encodedby any one of four different codons (CCT, CCC, CCA, CCG for proline;GCT, GCC, GCA, GCG for alanine; and GTT, GTC, GTA, GTG for valine). Someof these polynucleotides bear minimal homology to the nucleotidesequence of any native gene. Nonetheless, polynucleotides that vary dueto differences in codon usage are specifically contemplated by thepresent invention.

[0044] Polynucleotides, including target polynucleotides, may beprepared using any of a variety of techniques, which will be useful forthe preparation of specifically desired siRNA polynucleotides and forthe identification and selection of desirable sequences to be used insiRNA polynucleotides. For example, a polynucleotide may be amplifiedfrom cDNA prepared from a suitable cell or tissue type. Suchpolynucleotides may be amplified via polymerase chain reaction (PCR).For this approach, sequence-specific primers may be designed based onthe sequences provided herein and may be purchased or synthesized. Anamplified portion may be used to isolate a full-length gene, or adesired portion thereof, from a suitable library (e.g., human skeletalmuscle cDNA) using well known techniques. Within such techniques, alibrary (cDNA or genomic) is screened using one or more polynucleotideprobes or primers suitable for amplification. Preferably, a library issize-selected to include larger molecules. Random primed libraries mayalso be preferred for identifying 5′ and upstream regions of genes.Genomic libraries are preferred for obtaining introns and extending 5′sequences. Suitable sequences for a siRNA polynucleotide contemplated bythe present invention may also be selected from a library of siRNApolynucleotide sequences.

[0045] For hybridization techniques, a partial sequence may be labeled(e.g., by nick-translation or end-labeling with ³²P) using well knowntechniques. A bacterial or bacteriophage library may then be screened byhybridizing filters containing denatured bacterial colonies (or lawnscontaining phage plaques) with the labeled probe (see, e.g., Sambrook etal., Molecular Cloning: A Laboratory Manual, Cold Spring HarborLaboratories, Cold Spring Harbor, N.Y., 2001). Hybridizing colonies orplaques are selected and expanded, and the DNA is isolated for furtheranalysis. Clones may be analyzed to determine the amount of additionalsequence by, for example, PCR using a primer from the partial sequenceand a primer from the vector. Restriction maps and partial sequences maybe generated to identify one or more overlapping clones. A full-lengthcDNA molecule can be generated by ligating suitable fragments, usingwell known techniques.

[0046] Alternatively, numerous amplification techniques are known in theart for obtaining a full-length coding sequence from a partial cDNAsequence. Within such techniques, amplification is generally performedvia PCR. One such technique is known as “rapid amplification of cDNAends” or RACE. This technique involves the use of an internal primer andan external primer, which hybridizes to a polyA region or vectorsequence, to identify sequences that are 5′ and 3′ of a known sequence.Any of a variety of commercially available kits may be used to performthe amplification step. Primers may be designed using, for example,software well known in the art. Primers (or oligonucleotides for otheruses contemplated herein, including, for example, probes and antisenseoligonucleotides) are preferably 15, 16, 17, 18, 19, 20, 21, 22, 23, 24,25, 26, 27, 28, 29, 30, 31 or 32 nucleotides in length, have a GCcontent of at least 40% and anneal to the target sequence attemperatures of about 54° C. to 72° C. The amplified region may besequenced as described above, and overlapping sequences assembled into acontiguous sequence. Certain oligonucleotides contemplated by thepresent invention may, for some preferred embodiments, have lengths of18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33-35,35-40, 41-45, 46-50, 56-60, 61-70, 71-80, 81-90 or more nucleotides.

[0047] A number of specific siRNA polynucleotide sequences useful forinterfering with TCPTP polypeptide expression are presented in theExamples, the Drawings, and the Sequence Listing. SiRNA polynucleotidesmay generally be prepared by any method known in the art, including, forexample, solid phase chemical synthesis. Further, siRNAs may bechemically modified or conjugated to improve theur serum stabilityand/or delivery properties. Included as an aspect of the invention arethe siRNAs described herein wherein the ribose has been removedtherefrom. Modifications in a polynucleotide sequence may also beintroduced using standard mutagenesis techniques, such asoligonucleotide-directed site-specific mutagenesis. Alternatively, siRNApolynucleotide molecules may be generated by in vitro or in vivotranscription of suitable DNA sequences (e.g., polynucleotide sequencesencoding a PTP, or a desired portion thereof), provided that the DNA isincorporated into a vector with a suitable RNA polymerase promoter (suchas T7, U6, H1, or SP6). In addition, a siRNA polynucleotide may beadministered to a patient, as may be a DNA sequence (e.g., a recombinantnucleic acid construct as provided herein) that supports transcription(and optionally appropriate processing steps) such that a desired siRNAis generated in vivo.

[0048] Accordingly, a siRNA polynucleotide that is complementary to atleast a portion of a TCPTP coding sequence may be used to modulate geneexpression, or as a probe or primer. Identification of siRNApolynucleotide sequences and DNA encoding genes for their targeteddelivery involves techniques described herein with regard to TCPTP.Identification of such siRNA polynucleotide sequences and DNA encodinggenes for their targeted delivery involves techniques that are alsodescribed herein. As discussed above, siRNA polynucleotides exhibitdesirable stability characteristics and may, but need not, be furtherdesigned to resist degradation by endogenous nucleolytic enzymes byusing such linkages as phosphorothioate, methylphosphonate, sulfone,sulfate, ketyl, phosphorodithioate, phosphoramidate, phosphate esters,and other such linkages (see, e.g., Agrwal et al., Tetrahedron Lett.28:3539-3542 (1987); Miller et al., J. Am. Chem. Soc. 93:6657-6665(1971); Stec et al., Tetrahedron Lett. 26:2191-2194 (1985); Moody etal., Nucleic Acids Res. 12:4769-4782 (1989); Uznanski et al., NucleicAcids Res. (1989); Letsinger et al., Tetrahedron 40:137-143 (1984);Eckstein, Annu. Rev. Biochem. 54:367-402 (1985); Eckstein, Trends Biol.Sci. 14:97-100 (1989); Stein, In: Oligodeoxynucleotides. AntisenseInhibitors of Gene Expression, Cohen, ed., Macmillan Press, London, pp.97-117 (1989); Jager et al., Biochemistry 27:7237-7246 (1988)).

[0049] Any polynucleotide of the invention may be further modified toincrease stability in vivo. Possible modifications include, but are notlimited to, the addition of flanking sequences at the 5′ and/or 3′ ends;the use of phosphorothioate or 2′ O-methyl rather than phosphodiesterlinkages in the backbone; and/or the inclusion of nontraditional basessuch as inosine, queosine, and wybutosine and the like, as well asacetyl- methyl-, thio- and other modified forms of adenine, cytidine,guanine, thymine, and uridine.

[0050] Nucleotide sequences as described herein may be joined to avariety of other nucleotide sequences using established recombinant DNAtechniques. For example, a polynucleotide may be cloned into any of avariety of cloning vectors, including plasmids, phagemids, lambda phagederivatives, and cosmids. Vectors of particular interest includeexpression vectors, replication vectors, probe generation vectors, andsequencing vectors. In general, a suitable vector contains an origin ofreplication functional in at least one organism, convenient restrictionendonuclease sites, and one or more selectable markers. (See, e.g., WO01/96584; WO 01/29058; U.S. Pat. No. 6,326,193; U.S. Ser. No.2002/0007051). Other elements will depend upon the desired use, and willbe apparent to those having ordinary skill in the art. For example, theinvention contemplates the use of siRNA polynucleotide sequences in thepreparation of recombinant nucleic acid constructs including vectors forinterfering with the expression of a desired target polypeptide such asa TCPTP polypeptide in vivo; the invention also contemplates thegeneration of siRNA transgenic or “knock-out” animals and cells (e.g.,cells, cell clones, lines or lineages, or organisms in which expressionof one or more desired polypeptides (e.g., a target polypeptide) isfully or partially compromised). An siRNA polynucleotide that is capableof interfering with expression of a desired polypeptide (e.g., a targetpolypeptide) as provided herein thus includes any siRNA polynucleotidethat, when contacted with a subject or biological source as providedherein under conditions and for a time sufficient for target polypeptideexpression to take place in the absence of the siRNA polynucleotide,results in a statistically significant decrease (alternatively referredto as “knockdown” of expression) in the level of target polypeptideexpression that can be detected. Preferably the decrease is greater than10%, more preferably greater than 20%, more preferably greater than 30%,more preferably greater than 40%, 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%or 98% relative to the expression level of the polypeptide detected inthe absence of the siRNA, using conventional methods for determiningpolypeptide expression as known to the art and provided herein.Preferably, the presence of the siRNA polynucleotide in a cell does notresult in or cause any undesired toxic effects, for example, apoptosisor death of a cell in which apoptosis is not a desired effect of RNAinterference.

[0051] Within certain embodiments, siRNA polynucleotides may beformulated so as to permit entry into a cell of a mammal, and expressiontherein. Such formulations are particularly useful for therapeuticpurposes, as described below. Those having ordinary skill in the artwill appreciate that there are many ways to achieve expression of apolynucleotide in a target cell, and any suitable method may beemployed. For example, a polynucleotide may be incorporated into a viralvector using well known techniques (see also, e.g., U.S. Ser. No.2003/0068821). A viral vector may additionally transfer or incorporate agene for a selectable marker (to aid in the identification or selectionof transduced cells) and/or a targeting moiety, such as a gene thatencodes a ligand for a receptor on a specific target cell, to render thevector target specific. Targeting may also be accomplished using anantibody, by methods known to those having ordinary skill in the art.

[0052] Other formulations for therapeutic purposes include colloidaldispersion systems, such as macromolecule complexes, nanocapsules,microspheres, beads, and lipid-based systems including oil-in-wateremulsions, micelles, mixed micelles, and liposomes. A preferredcolloidal system for use as a delivery vehicle in vitro and in vivo is aliposome (i.e., an artificial membrane vesicle). The preparation and useof such systems is well known in the art.

[0053] Within other embodiments, one or more promoters may beidentified, isolated and/or incorporated into recombinant nucleic acidconstructs of the present invention, using standard techniques. Thepresent invention provides nucleic acid molecules comprising such apromoter sequence or one or more cis- or trans-acting regulatoryelements thereof. Such regulatory elements may enhance or suppressexpression of a siRNA. A 5′ flanking region may be generated usingstandard techniques, based on the genomic sequence provided herein. Ifnecessary, additional 5′ sequences may be generated using PCR-based orother standard methods. The 5′ region may be subcloned and sequencedusing standard methods. Primer extension and/or RNase protectionanalyses may be used to verify the transcriptional start site deducedfrom the cDNA.

[0054] To define the boundary of the promoter region, putative promoterinserts of varying sizes may be subcloned into a heterologous expressionsystem containing a suitable reporter gene without a promoter orenhancer. Suitable reporter genes may include genes encoding luciferase,beta-galactosidase, chloramphenicol acetyl transferase, secretedalkaline phosphatase, or the Green Fluorescent Protein gene (see, e.g.,Ui-Tei et al., FEBS Lett. 479:79-82 (2000). Suitable expression systemsare well known and may be prepared using well known techniques orobtained commercially. Internal deletion constructs may be generatedusing unique internal restriction sites or by partial digestion ofnon-unique restriction sites. Constructs may then be transfected intocells that display high levels of siRNA polynucleotide and/orpolypeptide expression. In general, the construct with the minimal 5′flanking region showing the highest level of expression of reporter geneis identified as the promoter. Such promoter regions may be linked to areporter gene and used to evaluate agents for the ability to modulatepromoter-driven transcription.

[0055] Once a functional promoter is identified, cis- and trans-actingelements may be located. Cis-acting sequences may generally beidentified based on homology to previously characterized transcriptionalmotifs. Point mutations may then be generated within the identifiedsequences to evaluate the regulatory role of such sequences. Suchmutations may be generated using site-specific mutagenesis techniques ora PCR-based strategy. The altered promoter is then cloned into areporter gene expression vector, as described above, and the effect ofthe mutation on reporter gene expression is evaluated.

[0056] In general, polypeptides and polynucleotides as described hereinare isolated. An “isolated” polypeptide or polynucleotide is one that isremoved from its original environment. For example, a naturallyoccurring protein is isolated if it is separated from some or all of thecoexisting materials in the natural system. Preferably, suchpolypeptides are at least about 90% pure, more preferably at least about95% pure and most preferably at least about 99% pure. A polynucleotideis considered to be isolated if, for example, it is cloned into a vectorthat is not a part of the natural environment. A “gene” includes thesegment of DNA involved in producing a polypeptide chain; it furtherincludes regions preceding and following the coding region “leader andtrailer,” for example promoter and/or enhancer and/or other regulatorysequences and the like, as well as intervening sequences (introns)between individual coding segments (exons).

[0057] As noted above, according to certain embodiments of the inventioncompositions and methods are provided that relate to altering or alteredTCPTP expression, and/or to a TCPTP associated disorder. A TCPTPassociated disorder includes any disease, disorder, condition, syndrome,pathologic or physiologic state, or the like, wherein at least oneundesirable deviation or departure from a physiological norm causes,correlates with, is accompanied by or results from an inappropriatealteration (i.e., a statistically significant change) to the structure,activity, function, expression level, physicochemical or hydrodynamicproperty, or stability of TCPTP or of a molecular component of abiological signal transduction pathway that comprises a TCPTP. Inpreferred embodiments the molecular component may be a protein, peptideor polypeptide, and in certain other preferred embodiments thealteration may be an altered level of TCPTP expression. In certain otherpreferred embodiments the alteration may be manifest as an atypical orunusual phosphorylation state of a protein under particular conditions,for example, hypophosphorylation or hyperphosphorylation of aphosphoprotein, wherein those familiar with the art will appreciate thatphosphorylated proteins typically comprise one or more phosphotyrosine,phosphoserine, or phosphothreonine residues.

[0058] TCPTP associated disorders therefore include, for example,diabetes mellitus, obesity, impaired glucose tolerance and othermetabolic disorders wherein alteration of TCPTP or of a TCPTP signalingpathway component is associated with the disorder. The effect of siRNAinterference with expression of a component in the signal transductionpathway induced by insulin, for example, may be evaluated by determiningthe level of tyrosine phosphorylation of insulin receptor beta (IR-β)and/or of the downstream signaling molecule PKB/Akt and/or of any otherdownstream polypeptide that may be a component of a particular signaltransduction pathway as provided herein. The invention is not intended,however, to be so limited and contemplates other disorders, such asJNK-associated disorders (e.g., cancer, cardiac hypertrophy, ischemia,diabetes, hyperglycemia-induced apoptosis, inflammation,neurodegenerative disorders), and other disorders associated withdifferent signal transduction pathways, for instance, cancer,autoimmunity, cellular proliferative disorders, neurodegenerativedisorders, and infectious diseases (see, e.g., Fukada et al., 2001 J.Biol. Chem. 276:25512; Tonks et al., 2001 Curr. Opin. Cell Biol. 13:182;Salmeen et al., 2000 Mol. Cell 6:1401; Hu et al., J. Neurochem.85:432-42 (2003); and references cited therein). Persons skilled in theart will be familiar with an array of criteria according to which it maybe recognized what are, for instance, biological, physiological,pathological and/or clinical signs and/or symptoms of TCPTP associatedand other disorders as provided herein.

[0059] In certain metabolic diseases or disorders, one or morebiochemical processes, which may be either anabolic or catabolic (e.g.,build-up or breakdown of substances, respectively), are altered (e.g.increased or decreased in a statistically significant manner) ormodulated (e.g., up- or down-regulated to a statistically significantdegree) relative to the levels at which they occur in a disease-free ornormal subject such as an appropriate control individual. The alterationmay result from an increase or decrease in a substrate, enzyme,cofactor, or any other component in any biochemical reaction involved ina particular process. Altered (i.e., increased or decreased in astatistically significant manner relative to a normal state) PTPactivity can underlie certain disorders and suggests a PTP role incertain metabolic diseases.

[0060] For example, disruption of the murine PTP1B gene homolog in aknock-out mouse model results in PTP1B^(−/−) mice exhibiting enhancedinsulin sensitivity, decreased levels of circulating insulin andglucose, and resistance to weight gain even on a high-fat diet, relativeto control animals having at least one functional PTP1B gene (Elcheblyet al., Science 283:1544 (1999)). Insulin receptor hyperphosphorylationhas also been detected in certain tissues of PTP1B deficient mice,consistent with a PTP1B contribution to the physiologic regulation ofinsulin and glucose metabolism (Id.). PTP-1B-deficient mice exhibitdecreased adiposity (reduced fat cell mass but not fat cell number),increased basal metabolic rate and energy expenditure, and enhancedinsulin-stimulated glucose utilization (Klaman et al., 2000 Mol. Cell.Biol. 20:5479). Additionally, altered PTP activity has been correlatedwith impaired glucose metabolism in other biological systems (e.g.,McGuire et al., Diabetes 40:939 (1991); Myerovitch et al., J. Clin.Invest. 84:976 (1989); Sredy et al., Metabolism 44:1074 (1995)),including PTP involvement in biological signal transduction via theinsulin receptor (see, e.g., WO 99/46268 and references cited therein).

[0061] As noted above, regulated tyrosine phosphorylation contributes tospecific pathways for biological signal transduction, including thoseassociated with cell division, cell survival, apoptosis, proliferationand differentiation, and “biological signal transduction pathways,” or“inducible signaling pathways” in the context of the present inventioninclude transient or stable associations or interactions among molecularcomponents involved in the control of these and similar processes incells. Depending on the particular pathway of interest, an appropriateparameter for determining induction of such pathway may be selected. Forexample, for signaling pathways associated with cell proliferation, avariety of well known methodologies are available for quantifyingproliferation, including, for example, incorporation of tritiatedthymidine into cellular DNA, monitoring of detectable (e.g.,fluorimetric or colorimetric) indicators of cellular respiratoryactivity (for example, conversion of the tetrazolium salts (yellow)3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) or3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulphophenyl)-2H-tetrazolium(MTS) to formazan dyes (purple) in metabolically active cells), or cellcounting, or the like. Similarly, in the cell biology arts, multipletechniques are known for assessing cell survival (e.g., vital dyes,metabolic indicators, etc.) and for determining apoptosis (for example,annexin V binding, DNA fragmentation assays, caspase activation, markeranalysis, e.g., poly(ADP-ribose) polymerase (PARP), etc.). Othersignaling pathways will be associated with particular cellularphenotypes, for example specific induction of gene expression (e.g.,detectable as transcription or translation products, or by bioassays ofsuch products, or as nuclear localization of cytoplasmic factors),altered (e.g., statistically significant increases or decreases) levelsof intracellular mediators (e.g., activated kinases or phosphatases,altered levels of cyclic nucleotides or of physiologically active ionicspecies, etc.), altered cell cycle profiles, or altered cellularmorphology, and the like, such that cellular responsiveness to aparticular stimulus as provided herein can be readily identified todetermine whether a particular cell comprises an inducible signalingpathway.

TCPTP

[0062] TCPTPs for use in the present invention include the following:(see, e.g., Andersen et al., 2001 Mol. Cell. Biol.; GenBank AccessionNos. M25393 (SEQ ID NOS: ______-______); M81478 (SEQ ID NO: ______);M80737 (SEQ ID NO: ______); M81477 (SEQ ID NOS: ______-______); X58828(SEQ ID NOS: ______-______); NM_(—)002828; and TC45 (e.g., NM_(—)080422(SEQ ID NOS: ______ and ______)). The invention also contemplates usingvariants or mutated forms of TCPTP, which may include a TCPTPpolypeptide that contains single nucleotide polymorphisms (SNPs), or mayinclude allelic forms.

[0063] Specific substitutions of individual amino acids throughintroduction of site-directed mutations are well-known and may be madeaccording to methodologies with which those having ordinary skill in theart will be familiar. The effects on catalytic activity of the resultingmutant PTP may be determined empirically by testing the resultingmodified protein for the preservation of the Km and reduction of Kcat toless than 1 per minute as provided herein and as previously disclosed(e.g., WO98/04712; Flint et al., 1997 Proc. Nat. Acad. Sci. USA94:1680). In addition, the effect on the ability of the resulting mutantPTP molecule to phosphorylate one or more tyrosine residues can also bedetermined empirically merely by testing such a mutant for the presenceof phosphotyrosine, as also provided herein, for example, followingexposure of the mutant to conditions in vitro or in vivo where it mayact as a phosphate acceptor for a protein tyrosine kinase.

[0064] In particular, portions of two TCPTP polypeptide sequences areregarded as “corresponding” amino acid sequences, regions, fragments orthe like, based on a convention of numbering one TCPTP sequenceaccording to amino acid position number, and then aligning the sequenceto be compared in a manner that maximizes the number of amino acids thatmatch or that are conserved residues, for example, that remain polar(e.g., D, E, K, R, H, S, T, N, Q), hydrophobic (e.g., A, P, V, L, I, M,F, W, Y) or neutral (e.g., C, G) residues at each position. Similarly, aDNA sequence encoding a candidate PTP that is to be mutated as providedherein, or a portion, region, fragment or the like, may correspond to aknown wildtype TCPTP-encoding DNA sequence according to a convention fornumbering nucleic acid sequence positions in the known wildtype TCPTPDNA sequence, whereby the candidate PTP DNA sequence is aligned with theknown TCPTP DNA such that at least 70%, preferably at least 80% and morepreferably at least 90% of the nucleotides in a given sequence of atleast 20 consecutive nucleotides of a sequence are identical. In certainpreferred embodiments, a candidate PTP DNA sequence is greater than 95%identical to a corresponding known TCPTP DNA sequence. In certainparticularly preferred embodiments, a portion, region or fragment of acandidate PTP DNA sequence is identical to a corresponding known TCPTPDNA sequence. As is well known in the art, an individual whose DNAcontains no irregularities (e.g., a common or prevalent form) in aparticular gene responsible for a given trait may be said to possess awildtype genetic complement (genotype) for that gene, while the presenceof irregularities known as mutations in the DNA for the gene, forexample, substitutions, insertions or deletions of one or morenucleotides, indicates a mutated or mutant genotype.

[0065] Modification of DNA may be performed by a variety of methods,including site-specific or site-directed mutagenesis of DNA encoding thepolypeptide of interest (e.g., a siRNA target polypeptide) and the useof DNA amplification methods using primers to introduce and amplifyalterations in the DNA template, such as PCR splicing by overlapextension (SOE). Site-directed mutagenesis is typically effected using aphage vector that has single- and double-stranded forms, such as M13phage vectors, which are well-known and commercially available. Othersuitable vectors that contain a single-stranded phage origin ofreplication may be used (see, e.g., Veira et al., Meth. Enzymol. 15:3,1987). In general, site-directed mutagenesis is performed by preparing asingle-stranded vector that encodes the protein of interest (e.g.,TCPTP). An oligonucleotide primer that contains the desired mutationwithin a region of homology to the DNA in the single-stranded vector isannealed to the vector followed by addition of a DNA polymerase, such asE. coli DNA polymerase I (Klenow fragment), which uses the doublestranded region as a primer to produce a heteroduplex in which onestrand encodes the altered sequence and the other the original sequence.Additional disclosure relating to site-directed mutagenesis may befound, for example, in Kunkel et al. (Methods in Enzymol. 154:367, 1987)and in U.S. Pat. Nos. 4,518,584 and 4,737,462. The heteroduplex isintroduced into appropriate bacterial cells, and clones that include thedesired mutation are selected. The resulting altered DNA molecules maybe expressed recombinantly in appropriate host cells to produce themodified protein.

[0066] SiRNAs of the invention may be fused to other nucleotidemolecules, or to polypeptides, in order to direct their delivery or toaccomplish other functions. Thus, for example, fusion proteinscomprising a siRNA oligonucleotide that is capable of specificallyinterfering with expression of TCPTP may comprise affinity tagpolypeptide sequences, which refers to polypeptides or peptides thatfacilitate detection and isolation of the such polypeptide via aspecific affinity interaction with a ligand. The ligand may be anymolecule, receptor, counterreceptor, antibody or the like with which theaffinity tag may interact through a specific binding interaction asprovided herein. Such peptides include, for example, poly-His or “FLAG®”or the like, e.g., the antigenic identification peptides described inU.S. Pat. No. 5,011,912 and in Hopp et al., (1988 Bio/Technology6:1204), or the XPRESS™ epitope tag (Invitrogen, Carlsbad, Calif.). Theaffinity sequence may be a hexa-histidine tag as supplied, for example,by a pBAD/His (Invitrogen) or a pQE-9 vector to provide for purificationof the mature polypeptide fused to the marker in the case of a bacterialhost, or, for example, the affinity sequence may be a hemagglutinin (HA)tag when a mammalian host, e.g., COS-7 cells, is used. The HA tagcorresponds to an antibody defined epitope derived from the influenzahemagglutinin protein (Wilson et al., 1984 Cell 37:767).

[0067] The present invention also relates to vectors and to constructsthat include or encode siRNA polynucleotides of the present invention,and in particular to “recombinant nucleic acid constructs” that includeany nucleic acid such as a DNA polynucleotide segment that may betranscribed to yield TCPTP polynucleotide-specific siRNA polynucleotidesaccording to the invention as provided above; to host cells which aregenetically engineered with vectors and/or constructs of the inventionand to the production of siRNA polynucleotides, polypeptides, and/orfusion proteins of the invention, or fragments or variants thereof, byrecombinant techniques. SiRNA sequences disclosed herein as RNApolynucleotides may be engineered to produce corresponding DNA sequencesusing well-established methodologies such as those described herein.Thus, for example, a DNA polynucleotide may be generated from any siRNAsequence described herein (including in the Sequence Listing), such thatthe present siRNA sequences will be recognized as also providingcorresponding DNA polynucleotides (and their complements). These DNApolynucleotides are therefore encompassed within the contemplatedinvention, for example, to be incorporated into the subject inventionrecombinant nucleic acid constructs from which siRNA may be transcribed.

[0068] According to the present invention, a vector may comprise arecombinant nucleic acid construct containing one or more promoters fortranscription of an RNA molecule, for example, the human U6 snRNApromoter (see, e.g., Miyagishi et al, Nat. Biotechnol. 20:497-500(2002); Lee et al., Nat. Biotechnol. 20:500-505 (2002); Paul et al.,Nat. Biotechnol. 20:505-508 (2002); Grabarek et al., BioTechniques34:73544 (2003); see also Sui et al., Proc. Natl. Acad. Sci. USA99:5515-20 (2002)). Each strand of a siRNA polynucleotide may betranscribed separately each under the direction of a separate promoterand then may hybridize within the cell to form the siRNA polynucleotideduplex. Each strand may also be transcribed from separate vectors (seeLee et al., supra). Alternatively, the sense and antisense sequencesspecific for a TCPTP sequence may be transcribed under the control of asingle promoter such that the siRNA polynucleotide forms a hairpinmolecule (Paul et al., supra). In such an instance, the complementarystrands of the siRNA specific sequences are separated by a spacer thatcomprises at least four nucleotides, but may comprise at least 5, 6, 7,8, 9, 10, 11, 12, 14, 16, 94 18 nucleotides or more nucleotides asdescribed herein. In addition, siRNAs transcribed under the control of aU6 promoter that form a hairpin may have a stretch of about foururidines at the 3′ end that act as the transcription termination signal(Miyagishi et al., supra; Paul et al., supra). By way of illustration,if the target sequence is 19 nucleotides, the siRNA hairpinpolynucleotide (beginning at the 5′ end) has a 1 9-nucleotide sensesequence followed by a spacer (which as two uridine nucleotides adjacentto the 3′ end of the 19-nucleotide sense sequence), and the spacer islinked to a 19 nucleotide antisense sequence followed by a 4-uridineterminator sequence, which results in an overhang. SiRNA polynucleotideswith such overhangs effectively interfere with expression of the targetpolypeptide (see id.). A recombinant construct may also be preparedusing another RNA polymerase III promoter, the H1 RNA promoter, that maybe operatively linked to siRNA polynucleotide specific sequences, whichmay be used for transcription of hairpin structures comprising the siRNAspecific sequences or separate transcription of each strand of a siRNAduplex polynucleotide (see, e.g., Brummelkamp et al., Science 296:550-53(2002); Paddison et al., supra). DNA vectors useful for insertion ofsequences for transcription of an siRNA polynucleotide include pSUPERvector (see, e.g., Brummelkamp et al., supra); pAV vectors derived frompCWRSVN (see, e.g., Paul et al., supra); and pIND (see, e.g., Lee etal., supra), or the like.

[0069] TCPTP polypeptides can be expressed in mammalian cells, yeast,bacteria, or other cells under the control of appropriate promoters,providing ready systems for evaluation of siRNA polynucleotides that arecapable of interfering with polypeptide expression as provided herein.Appropriate cloning and expression vectors for use with prokaryotic andeukaryotic hosts are described, for example, by Sambrook, et al.,Molecular Cloning: A Laboratory Manual, Third Edition, Cold SpringHarbor, N.Y., (2001).

[0070] Generally, recombinant expression vectors for use in thepreparation of recombinant nucleic acid constructs or vectors of theinvention will include origins of replication and selectable markerspermitting transformation of the host cell, e.g., the ampicillinresistance gene of E. coli and S. cerevisiae TRP1 gene, and a promoterderived from a highly-expressed gene to direct transcription of adownstream structural sequence (e.g., a siRNA polynucleotide sequence).Such promoters can be derived from operons encoding glycolytic enzymessuch as 3-phosphoglycerate kinase (PGK), α-factor, acid phosphatase, orheat shock proteins, among others. For PTP polypeptide expression(including PTP fusion proteins and substrate trapping mutant PTPs), andfor other expression of other polypeptides of interest, the heterologousstructural sequence is assembled in appropriate phase with translationinitiation and termination sequences. Optionally, the heterologoussequence can encode a fusion protein including an N-terminalidentification peptide imparting desired characteristics, e.g.,stabilization or simplified purification of expressed recombinantproduct.

[0071] Useful expression constructs for bacterial use are constructed byinserting into an expression vector a structural DNA sequence encoding adesired siRNA polynucleotide, together with suitable transcriptioninitiation and termination signals in operable linkage, for example,with a functional promoter. The construct may comprise one or morephenotypic selectable markers and an origin of replication to ensuremaintenance of the vector construct and, if desirable, to provideamplification within the host. Suitable prokaryotic hosts fortransformation include E. coli, Bacillus subtilis, Salmonellatyphimurium and various species within the genera Pseudomonas,Sireptomyces, and Staphylococcus, although others may also be employedas a matter of choice. Any other plasmid or vector may be used as longas they are replicable and viable in the host.

[0072] As a representative but nonlimiting example, useful expressionvectors for bacterial use can comprise a selectable marker and bacterialorigin of replication derived from commercially available plasmidscomprising genetic elements of the well known cloning vector pBR322(ATCC 37017). Such commercial vectors include, for example, pKK223-3(Pharmacia Fine Chemicals, Uppsala, Sweden) and GEM1 (Promega Biotec,Madison, Wis., USA). These pBR322 “backbone” sections are combined withan appropriate promoter and the structural sequence to be expressed.

[0073] Following transformation of a suitable host strain and growth ofthe host strain to an appropriate cell density, the selected promoter,if it is a regulated promoter as provided herein, is induced byappropriate means (e.g., temperature shift or chemical induction) andcells are cultured for an additional period. Cells are typicallyharvested by centrifugation, disrupted by physical or chemical means,and the resulting crude extract retained for further purification.Microbial cells employed in expression of proteins can be disrupted byany convenient method, including freeze-thaw cycling, sonication,mechanical disruption, or use of cell lysing agents; such methods arewell know to those skilled in the art.

[0074] Thus, for example, the nucleic acids of the invention asdescribed herein (e.g., DNA sequences from which siRNA may betranscribed) may be included in any one of a variety of expressionvector constructs as a recombinant nucleic acid construct for expressinga TCPTP polynucleotide-specific siRNA polynucleotide as provided herein.Such vectors and constructs include chromosomal, nonchromosomal andsynthetic DNA sequences, e.g., derivatives of SV40; bacterial plasmids;phage DNA; baculovirus; yeast plasmids; vectors derived fromcombinations of plasmids and phage DNA, viral DNA, such as vaccinia,adenovirus, fowl pox virus, and pseudorabies. However, any other vectormay be used for preparation of a recombinant nucleic acid construct aslong as it is replicable and viable in the host.

[0075] The appropriate DNA sequence(s) may be inserted into the vectorby a variety of procedures. In general, the DNA sequence is insertedinto an appropriate restriction endonuclease site(s) by procedures knownin the art. Standard techniques for cloning, DNA isolation,amplification and purification, for enzymatic reactions involving DNAligase, DNA polymerase, restriction endonucleases and the like, andvarious separation techniques are those known and commonly employed bythose skilled in the art. A number of standard techniques are described,for example, in Ausubel et al. (1993 Current Protocols in MolecularBiology, Greene Publ. Assoc. Inc. & John Wiley & Sons, Inc., Boston,Mass.); Sambrook et al. (2001 Molecular Cloning, Third Ed., Cold SpringHarbor Laboratory, Plainview, N.Y.); Maniatis et al. (1982 MolecularCloning, Cold Spring Harbor Laboratory, Plainview, N.Y.); and elsewhere.

[0076] The DNA sequence in the expression vector is operatively linkedto at least one appropriate expression control sequences (e.g., apromoter or a regulated promoter) to direct mRNA synthesis.Representative examples of such expression control sequences include LTRor SV40 promoter, the E. coli lac or trp, the phage lambda P_(L)promoter and other promoters known to control expression of genes inprokaryotic or eukaryotic cells or their viruses. Promoter regions canbe selected from any desired gene using CAT (chloramphenicoltransferase) vectors or other vectors with selectable markers. Twoappropriate vectors are pKK232-8 and pCM7. Particular named bacterialpromoters include lac, lacZ, T3, T7, gpt, lambda P_(R), P_(L) and trp.Eukaryotic promoters include CMV immediate early, HSV thymidine kinase,early and late SV40, LTRs from retrovirus, and mouse metallothionein-I.Selection of the appropriate vector and promoter is well within thelevel of ordinary skill in the art, and preparation of certainparticularly preferred recombinant expression constructs comprising atleast one promoter or regulated promoter operably linked to a nucleicacid encoding a TCPTP polypeptide is described herein.

[0077] As noted above, in certain embodiments the vector may be a viralvector such as a retroviral vector. For example, retroviruses from whichthe retroviral plasmid vectors may be derived include, but are notlimited to, Moloney Murine Leukemia Virus, spleen necrosis virus,retroviruses such as Rous Sarcoma Virus, Harvey Sarcoma virus, avianleukosis virus, gibbon ape leukemia virus, human immunodeficiency virus,adenovirus, Myeloproliferative Sarcoma Virus, and mammary tumor virus.

[0078] The viral vector includes one or more promoters. Suitablepromoters which may be employed include, but are not limited to, theretroviral LTR; the SV40 promoter; and the human cytomegalovirus (CMV)promoter described in Miller, et al., Biotechniques 7:980-990 (1989), orany other promoter (e.g., cellular promoters such as eukaryotic cellularpromoters including, but not limited to, the histone, pol III, andβ-actin promoters). Other viral promoters that may be employed include,but are not limited to, adenovirus promoters, thymidine kinase (TK)promoters, and B19 parvovirus promoters. The selection of a suitablepromoter will be apparent to those skilled in the art from the teachingscontained herein, and may be from among either regulated promoters orpromoters as described above.

[0079] The retroviral plasmid vector is employed to transduce packagingcell lines to form producer cell lines. Examples of packaging cellswhich may be transfected include, but are not limited to, the PE501,PA317, ψ-2, ψ-AM, PA12, T19-14X, VT-19-17-H2, ψCRE, ψCRIP, GP+E-86,GP+envAm12, and DAN cell lines as described in Miller, Human GeneTherapy, 1:5-14 (1990), which is incorporated herein by reference in itsentirety. The vector may transduce the packaging cells through any meansknown in the art. Such means include, but are not limited to,electroporation, the use of liposomes, and calcium phosphateprecipitation. In one alternative, the retroviral plasmid vector may beencapsulated into a liposome, or coupled to a lipid, and thenadministered to a host.

[0080] The producer cell line generates infectious retroviral vectorparticles that include the nucleic acid sequence(s) encoding the TCPTPpolypeptide and variants and fusion proteins thereof. Such retroviralvector particles then may be employed, to transduce eukaryotic cells,either in vitro or in vivo. The transduced eukaryotic cells will expressthe nucleic acid sequence(s) encoding the siRNA polynucleotide that iscapable of specifically interfering with expression of a polypeptide orfusion protein. Eukaryotic cells which may be transduced include, butare not limited to, embryonic stem cells, embryonic carcinoma cells, aswell as hematopoietic stem cells, hepatocytes, fibroblasts, myoblasts,keratinocytes, endothelial cells, bronchial epithelial cells and variousother culture-adapted cell lines.

[0081] In another aspect, the present invention relates to host cellscontaining the above described recombinant TCPTP expression constructs.Host cells are genetically engineered (transduced, transformed ortransfected) with the vectors and/or expression constructs of thisinvention that may be, for example, a cloning vector, a shuttle vector,or an expression construct. The vector or construct may be, for example,in the form of a plasmid, a viral particle, a phage, etc. The engineeredhost cells can be cultured in conventional nutrient media modified asappropriate for activating promoters, selecting transformants oramplifying particular genes such as genes encoding siRNA polynucleotidesor fusion proteins thereof. The culture conditions for particular hostcells selected for expression, such as temperature, pH and the like,will be readily apparent to the ordinarily skilled artisan.

[0082] The host cell can be a higher eukaryotic cell, such as amammalian cell, or a lower eukaryotic cell, such as a yeast cell, or thehost cell can be a prokaryotic cell, such as a bacterial cell.Representative examples of appropriate host cells according to thepresent invention include, but need not be limited to, bacterial cells,such as E. coli, Streptomyces, Salmonella typhimurium; fungal cells,such as yeast; insect cells, such as Drosophila S2 and Spodoptera Sf9;animal cells, such as CHO, COS or 293 cells; adenoviruses; plant cells,or any suitable cell already adapted to in vitro propagation or soestablished de novo. The selection of an appropriate host is deemed tobe within the scope of those skilled in the art from the teachingsherein.

[0083] Various mammalian cell culture systems can also be employed toproduce siRNA polynucleotides from recombinant nucleic acid constructsof the present invention. The invention is therefore directed in part toa method of producing a siRNA polynucleotide, by culturing a host cellcomprising a recombinant nucleic acid construct that comprises at leastone promoter operably linked to a nucleic acid sequence encoding a siRNApolynucleotide specific for a TCPTP polypeptide. In certain embodiments,the promoter may be a regulated promoter as provided herein, for examplea tetracylcine-repressible promoter. In certain embodiments therecombinant expression construct is a recombinant viral expressionconstruct as provided herein. Examples of mammalian expression systemsinclude the COS-7 lines of monkey kidney fibroblasts, described byGluzman, Cell 23:175 (1981), and other cell lines capable of expressinga compatible vector, for example, the C127, 3T3, CHO, HeLa, HEK, and BHKcell lines. Mammalian expression vectors will comprise an origin ofreplication, a suitable promoter and enhancer, and also any necessaryribosome binding sites, polyadenylation site, splice donor and acceptorsites, transcriptional termination sequences, and 5′ flankingnontranscribed sequences, for example as described herein regarding thepreparation of recombinant siRNA polynucleotide constructs. DNAsequences derived from the SV40 splice, and polyadenylation sites may beused to provide the required nontranscribed genetic elements.Introduction of the construct into the host cell can be effected by avariety of methods with which those skilled in the art will be familiar,including but not limited to, for example, liposomes including cationicliposomes, calcium phosphate transfection, DEAE-Dextran mediatedtransfection, or electroporation (Davis et al., 1986 Basic Methods inMolecular Biology), or other suitable technique.

[0084] The expressed recombinant siRNA polynucleotides may be useful inintact host cells; in intact organelles such as cell membranes,intracellular vesicles or other cellular organelles; or in disruptedcell preparations including but not limited to cell homogenates orlysates, microsomes, uni- and multilamellar membrane vesicles or otherpreparations. Alternatively, expressed recombinant siRNA polynucleotidescan be recovered and purified from recombinant cell cultures by methodsincluding ammonium sulfate or ethanol precipitation, acid extraction,anion or cation exchange chromatography, phosphocellulosechromatography, hydrophobic interaction chromatography, affinitychromatography, hydroxylapatite chromatography and lectinchromatography. Finally, high performance liquid chromatography (HPLC)can be employed for final purification steps.

Samples

[0085] According to the present invention, a method is provided forinterfering with expression of a PTP polypeptide such as TCPTP, or avariant thereof, as provided herein. A method is also provided forinterfering with expression of a TCPTP polypeptide, comprisingcontacting a siRNA polynucleotide with a cell that is capable ofexpressing TCPTP, typically in a biological sample or in a subject orbiological source. A “sample” as used herein refers to a biologicalsample containing at least one desired target protein, and may beprovided by obtaining a blood sample, biopsy specimen, tissue explant,organ culture or any other tissue or cell preparation from a subject ora biological source. A sample may further refer to a tissue or cellpreparation in which the morphological integrity or physical state hasbeen disrupted, for example, by dissection, dissociation,solubilization, fractionation, homogenization, biochemical or chemicalextraction, pulverization, lyophilization, sonication or any other meansfor processing a sample derived from a subject or biological source. Incertain preferred embodiments, the sample is a cell that comprises atleast one TCPTP polypeptide, and in certain particularly preferredembodiments the cell comprises an inducible biological signalingpathway, at least one component of which is TCPTP. In particularlypreferred embodiments the cell is a mammalian cell, for example, Rat-1fibroblasts, COS cells, CHO cells, HEK-293 cells, HepG2, HII4E-C3, L6,and 3T3-L1, or other well known model cell lines, which are availablefrom the American Type Culture Collection (ATCC, Manassas, Va.). Inother preferred embodiments, the cell line is derived from TCPTPknockout animals and which may be transfected with human insulinreceptor (HIR), for example, 1 BKO mouse embryo fibroblasts.

[0086] In certain other preferred embodiments the sample is a cell thatincludes, for example, a cell line that is derived from a tumor cell.The cell line may be a primary tumor cell line, that is, a cell lineprepared directly from a tumor sample removed from a human or anon-human animal. Alternatively, the cell line may be one of severalestablished tumor cell lines known in the art, including but not limitedto MCF7, T47D, SW620, HS578T, MDA-MB-435, MDA MB 231, HCT-116, HT-29,HeLa, Raji, Ramos, and the like (see ATCC collection).

[0087] The subject or biological source may be a human or non-humananimal, a primary cell culture or culture adapted cell line includingbut not limited to genetically engineered cell lines that may containchromosomally integrated or episomal recombinant nucleic acid sequences,immortalized or immortalizable cell lines, somatic cell hybrid celllines, differentiated or differentiatable cell lines, transformed celllines and the like. Optionally, in certain situations it may bedesirable to treat cells in a biological sample with hydrogen peroxideand/or with another agent that directly or indirectly promotes reactiveoxygen species (ROS) generation, including biological stimuli asdescribed herein; in certain other situations it may be desirable totreat cells in a biological sample with a ROS scavenger, such asN-acetyl cysteine (NAC) or superoxide dismutase (SOD) or other ROSscavengers known in the art; in other situations cellular glutathione(GSH) may be depleted by treating cells with L-buthionine-SR-sulfoximine(Bso); and in other circumstances cells may be treated with pervanadateto enrich the sample in tyrosine phosphorylated proteins. Other meansmay also be employed to effect an increase in the population of tyrosinephosphorylated proteins present in the sample, including the use of asubject or biological source that is a cell line that has beentransfected with at least one gene encoding a protein tyrosine kinase.

[0088] Additionally or alternatively, a biological signaling pathway maybe induced in subject or biological source cells by contacting suchcells with an appropriate stimulus, which may vary depending upon thesignaling pathway under investigation, whether known or unknown. Forexample, a signaling pathway that, when induced, results in proteintyrosine phosphorylation and/or protein tyrosine dephosphorylation maybe stimulated in subject or biological source cells using any one ormore of a variety of well known methods and compositions known in theart to stimulate protein tyrosine kinase (PTK) and/or PTP activity.These stimuli may include, without limitation, exposure of cells tocytokines, growth factors, hormones, peptides, small molecule mediators,cell stressors (e.g., ultraviolet light; temperature shifts; osmoticshock; ROS or a source thereof, such as hydrogen peroxide, superoxide,ozone, etc. or any agent that induces or promotes ROS production (see,e.g., Halliwell and Gutteridge, Free Radicals in Biology and Medicine(3^(rd) Ed.) 1999 Oxford University Press, Oxford, UK); heavy metals;alcohol) or other agents that induce PTK-mediated protein tyrosinephosphorylation and/or PTP-mediated phosphoprotein tyrosinedephosphorylation. Such agents may include, for example, interleukins(e.g., IL-1, IL-3), interferons (e.g., IFN-γ), human growth hormone,insulin, epidermal growth factor (EGF), platelet derived growth factor(PDGF), granulocyte colony stimulating factor (G-CSF),granulocyte-megakaryocyte colony stimulating factor (GM-CSF),transforming growth factor (e.g., TGF-β1), tumor necrosis factor (e.g.,TNF-α) and fibroblast growth factor (FGF; e.g., basic FGF (bFGF)), anyagent or combination of agents capable of triggering T lymphocyteactivation via the T cell receptor for antigen (TCR; TCR-inducing agentsmay include superantigens, specifically recognized antigens and/orMHC-derived peptides, MHC peptide tetramers (e.g., Altman et al., 1996Science 274:94-96); TCR-specific antibodies or fragments or derivativesthereof), lectins (e.g., PHA, PWM, ConA, etc.), mitogens, G-proteincoupled receptor agonists such as angiotensin-2, thrombin, thyrotropin,parathyroid hormone, lysophosphatidic acid (LPA),sphingosine-1-phosphate, serotonin, endothelin, acetylcholine, plateletactivating factor (PAF) or bradykinin, as well as other agents withwhich those having ordinary skill in the art will be familiar (see,e.g., Rhee et al., [online] 10 Oct. 2000 Science's stke,Internet:URL<www.stke.org/cgl/content/full/ OC_sigtrans;2000/53/pe1>),and references cited therein).

[0089] As noted above, regulated tyrosine phosphorylation contributes tospecific pathways for biological signal transduction, including thoseassociated with cell division, cell survival, apoptosis, proliferationand differentiation, and “inducible signaling pathways” in the contextof the present invention include transient or stable associations orinteractions among molecular components involved in the control of theseand similar processes in cells. Depending on the particular pathway ofinterest, an appropriate parameter for determining induction of suchpathway may be selected. For example, for signaling pathways associatedwith cell proliferation, a variety of well known methodologies areavailable for quantifying proliferation, including, for example,incorporation of tritiated thymidine into cellular DNA, monitoring ofdetectable (e.g., fluorimetric or colorimetric) indicators of cellularrespiratory activity, (e.g., MTT assay) or cell counting, or the like.Similarly, in the cell biology arts there are known multiple techniquesfor assessing cell survival (e.g., vital dyes, metabolic indicators,etc.) and for determining apoptosis (e.g., annexin V binding, DNAfragmentation assays, caspase activation, PARP cleavage, etc.). Othersignaling pathways will be associated with particular cellularphenotypes, for example specific induction of gene expression (e.g.,detectable as transcription or translation products, or by bioassays ofsuch products, or as nuclear localization of cytoplasmic factors),altered (e.g., statistically significant increases or decreases) levelsof intracellular mediators (e.g., activated kinases or phosphatases,altered levels of cyclic nucleotides or of physiologically active ionicspecies, etc.), altered cell cycle profiles, or altered cellularmorphology, and the like, such that cellular responsiveness to aparticular stimulus as provided herein can be readily identified todetermine whether a particular cell comprises an inducible signalingpathway.

[0090] In preferred embodiments, a TCPTP substrate may be any naturallyor non-naturally occurring phosphorylated peptide, polypeptide orprotein that can specifically bind to and/or be dephosphorylated by aTCPTP polypeptide.

[0091] Identification and selection of TCPTP substrates as providedherein, for use in the present invention, may be performed according toprocedures with which those having ordinary skill in the art will befamiliar, or may, for example, be conducted according to the disclosuresof WO 00/75339, U.S. application Ser. No. 09/334,575, or U.S.application Ser. No. 10/366,547, and references cited therein. Thephosphorylated protein/PTP complex may be isolated, for example, byconventional isolation techniques as described in U.S. Pat. No.5,352,660, including salting out, chromatography, electrophoresis, gelfiltration, fractionation, absorption, polyacrylamide gelelectrophoresis, agglutination, combinations thereof or otherstrategies. TCPTP substrates that are known may also be preparedaccording to well known procedures that employ principles of molecularbiology and/or peptide synthesis (e.g., Ausubel et al., CurrentProtocols in Molecular Biology, Greene Publ. Assoc. Inc. & John Wiley &Sons, Inc., Boston, Mass. (1993); Sambrook et al., Molecular Cloning,Third Ed., Cold Spring Harbor Laboratory, Plainview, N.Y. (2001); Fox,Molec. Biotechnol. 3:249 (1995); Maeji et al., Pept. Res. 8:33 (1995)).

[0092] The TCPTP substrate peptides of the present invention maytherefore be derived from TCPTP substrate proteins, polypeptides andpeptides as provided herein having amino acid sequences that areidentical or similar to tyrosine phosphorylated TCPTP substratesequences known in the art. For example by way of illustration and notlimitation, peptide sequences derived from the known TCPTP substrateproteins referred to above are contemplated for use according to theinstant invention, as are peptides having at least 70% similarity(preferably 70% identity), more preferably 80% similarity (morepreferably 80% identity), more preferably 90% similarity (morepreferably 90% identity) and still more preferably 95% similarity (stillmore preferably 95% identity) to the polypeptides described inreferences cited herein and in the Examples and to portions of suchpolypeptides as disclosed herein. As known in the art “similarity”between two polypeptides is determined by comparing the amino acidsequence and conserved amino acid substitutes thereto of the polypeptideto the sequence of a second polypeptide (e.g., using GENEWORKS, Align orthe BLAST algorithm, or another algorithm, as described above).

[0093] In certain preferred embodiments of the present invention, thesiRNA polynucleotide and/or the TCPTP substrate is detectably labeled,and in particularly preferred embodiments the siRNA polynucleotideand/or PTP substrate is capable of generating a radioactive or afluorescent signal. The siRNA polynucleotide and/or PTP substrate can bedetectably labeled by covalently or non-covalently attaching a suitablereporter molecule or moiety, for example a radionuclide such as ³²P(e.g., Pestka et al., 1999 Protein Expr. Purif. 17:203-14), aradiohalogen such as iodine [¹²⁵I or ¹³¹I] (e.g., Wilbur, 1992Bioconjug. Chem. 3:433-70), or tritium [³H]; an enzyme; or any ofvarious luminescent (e.g., chemiluminescent) or fluorescent materials(e.g., a fluorophore) selected according to the particular fluorescencedetection technique to be employed, as known in the art and based uponthe present disclosure. Fluorescent reporter moieties and methods forlabeling siRNA polynucleotides and/or PTP substrates as provided hereincan be found, for example in Haugland (1996 Handbook of FluorescentProbes and Research Chemicals—Sixth Ed., Molecular Probes, Eugene,Oreg.; 1999 Handbook of Fluorescent Probes and ResearchChemicals—Seventh Ed., Molecular Probes, Eugene, Oreg., [Internet]<:http://www.probes.com/lit/>) and in references cited therein.Particularly preferred for use as such a fluorophore in the subjectinvention methods are fluorescein, rhodamine, Texas Red, AlexaFluor-594,AlexaFluor-488, Oregon Green, BODIPY-FL, umbelliferone,dichlorotriazinylamine fluorescein, dansyl chloride, phycoerythrin orCy-5. Examples of suitable enzymes include, but are not limited to,horseradish peroxidase, biotin, alkaline phosphatase, β-galactosidaseand acetylcholinesterase. Appropriate luminescent materials includeluminol, and suitable radioactive materials include radioactivephosphorus [³²P]. In certain other preferred embodiments of the presentinvention, a detectably labeled siRNA polynucleotide comprises amagnetic particle, for example a paramagnetic or a diamagnetic particleor other magnetic particle or the like (preferably a microparticle)known to the art and suitable for the intended use. Without wishing tobe limited by theory, according to certain such embodiments there isprovided a method for selecting a cell that has bound, adsorbed,absorbed, internalized or otherwise become associated with a siRNApolynucleotide that comprises a magnetic particle. For example,selective isolation of a population or subpopulation of cells containingone or more TCPTP-specific siRNA polynucleotide-magnetic particleconjugates may offer certain advantages in the further characterizationor regulation of PTP signaling pathways.

[0094] In certain embodiments of the present invention, particularTCPTP-specific siRNA polynucleotides of interest may be identified bycontacting a candidate siRNA polynucleotide with a sample comprising acell that comprises a TCPTP gene and that is capable of TCPTP genetranscription or expression (e.g., translation), under conditions andfor a time sufficient to detect TCPTP gene transcription or expression,and comparing TCPTP transcription levels, TCPTP polypeptide expressionand/or TCPTP functional expression (e.g., TCPTP catalytic activity) inthe absence and presence of the candidate siRNA polynucleotide.Preferably TCPTP transcription or expression is decreased in thepresence of the siRNA polynucleotide, thereby providing an alternativeto TCPTP active site directed approaches to modulating TCPTP activity.(The invention need not be so limited, however, and contemplates otherembodiments wherein one or more transcription and/or expression levelsof a signal transduction component other than that which is specificallytargeted by the siRNA may be increased in the presence of a certainTCPTP-specific siRNA polynucleotide. By way of non-limiting theory, suchan increase may result from a cellular compensatory mechanism that isinduced as a result of the siRNA.)

[0095] For a cell that expresses TCPTP and comprises an insulinreceptor, such as IR-β, and the siRNA polynucleotide effects an increasein insulin receptor phosphorylation, presumably (and according tonon-binding theory) by decreasing TCPTP levels through interference withTCPTP expression. Methods for determining insulin receptorphosphorylation are known in the art (e.g., Cheatham et al., 1995Endocr. Rev. 16:117-142) and are described in greater detail below. Incertain other further embodiments wherein the cell comprises an insulinreceptor, any of a variety of cellular insulin responses may bemonitored according to art-established methodologies, including but notlimited to glucose uptake (e.g., Elchebly et al., 1999 Science 283:1544;McGuire et al., 1991 Diabetes 40:939; Myerovitch et al., 1989 J. Clin.Invest. 84:976; Sredy et al. 1995 Metabolism 44:1074; WO 99/46268);glycogen synthesis (e.g., Berger et al., 1998 Anal. Biochem. 261:159),Glut4 recruitment to a plasma membrane (Robinson et al., 1992 J. CellBiol. 117:1181); liver transcription events, or amino acid import (Hydeet al., 2002 J. Biol. Chem. 277:13628-34 (2002)). In certain otherfurther embodiments wherein the cell comprises an insulin receptor,cellular insulin responses that may be monitored include MAP kinasephosphorylation, AKT phosphorylation, and other insulin-stimulatedphosphorylation events downstream of the insulin receptor, such as PI3kinase, perk, pSTAT5, and IRS1, and inhibition of phosphoenolpyruvatecarboxykinase transcription (Forest et al., 1990 Molec. Endocrinol.4:1302), phosphatidylinositoltriphosphate kinase activation (Endeman etal., 1990 J. Biol. Chem. 265:396), lipogenesis (Moody et al., 1974 Horm.Metab. Res. 6:12), lipolysis (Hess et al., 1991 J. Cell. Biochem.45:374), TYK2 dephosphorylation and JAK2 (see GenBank Nos. NM_(—)004972,AF058925, AF005216, NM_(—)031514, and NM_(—)008261) dephosphorylation(Myers et al., 2001 J. Biol. Chem. 276:47771), interferon-stimulatedpSTAT1 and pSTAT3, and EGF or PDGRF phosphorylation (Ullrich et al.,1990 Cell 61:203). In addition, phosphorylation of the insulin receptor,such as at positions tyr1162/tyr1163 and at position tyr972, may bedetected with anti-phosphotyrosine antibodies that are site-specific fortyr1162/tyr1163 or tyr972.

[0096] TCPTP activity may also be measured in whole cells transfectedwith a reporter gene whose expression is dependent upon the activationof an appropriate substrate. For example, appropriate cells (i.e., cellsthat are capable of expressing TCPTP and that have been transfected witha TCPTP-specific siRNA polynucleotide that is either known or suspectedof being capable of interfering with TCPTP polypeptide expression) maybe transfected with a substrate-dependent promoter linked to a reportergene. In such a system, expression of the reporter gene (which may bereadily detected using methods well known to those of ordinary skill inthe art) depends upon activation of substrate. Dephosphorylation ofsubstrate may be detected based on a decrease in reporter activity.Candidate siRNA polynucleotides specific for TCPTP may be added to sucha system, as described above, to evaluate their effect on TCPTPactivity.

[0097] Within other aspects, the present invention provides animalmodels in which an animal, by virtue of introduction of an appropriateTCPTP-specific siRNA polynucleotide, for example, as a transgene, doesnot express (or expresses a significantly reduced amount of) afunctional TCPTP. Such animals may be generated, for example, usingstandard homologous recombination strategies, or alternatively, forinstance, by oocyte microinjection with a plasmid comprising thesiRNA-encoding sequence that is regulated by a suitable promoter (e.g.,ubiquitous or tissue-specific) followed by implantation in a surrogatemother. Animal models generated in this manner may be used to studyactivities of PTP signaling pathway components and modulating agents invivo.

[0098] Therapeutic Methods

[0099] One or more siRNA polynucleotides capable of interfering withTCPTP polypeptide expression and identified according to theabove-described methods may also be used to modulate (e.g., inhibit orpotentiate) TCPTP activity in a patient. As used herein, a “patient” maybe any mammal, including a human, and may be afflicted with a conditionassociated with undesired TCPTP activity or may be free of detectabledisease. Accordingly, the treatment may be of an existing disease or maybe prophylactic. Conditions associated with signal transduction and/orTCPTP activity include any disorder associated with cell proliferation,including cancer, graft-versus-host disease (GVHD), autoimmune diseases,allergy or other conditions in which unregulated TCPTP activity may beinvolved, metabolic diseases (including e.g., diabetes mellitus,obesity, impaired glucose tolerance), abnormal cell growth orproliferation and cell cycle abnormalities.

[0100] For administration to a patient, one or more specific siRNApolynucleotides, either alone, with or without chemical modification orremoval of ribose, or comprised in an appropriate vector as describedherein (e.g., including a vector which comprises a DNA sequence fromwhich a specific siRNA can be transcribed) are generally formulated as apharmaceutical composition. A pharmaceutical composition may be asterile aqueous or non-aqueous solution, suspension or emulsion, whichadditionally comprises a physiologically acceptable carrier (i.e., anon-toxic material that does not interfere with the activity of theactive ingredient). Such compositions may be in the form of a solid,liquid or gas (aerosol). Alternatively, compositions of the presentinvention may be formulated as a lyophilizate or compounds may beencapsulated within liposomes using well known technology.Pharmaceutical compositions within the scope of the present inventionmay also contain other components, which may be biologically active orinactive. Such components include, but are not limited to, buffers(e.g., neutral buffered saline or phosphate buffered saline),carbohydrates (e.g., glucose, mannose, sucrose or dextrans), mannitol,proteins, polypeptides or amino acids such as glycine, antioxidants,chelating agents such as EDTA or glutathione, stabilizers, dyes,flavoring agents, and suspending agents and/or preservatives.

[0101] Any suitable carrier known to those of ordinary skill in the artmay be employed in the pharmaceutical compositions of the presentinvention. Carriers for therapeutic use are well known, and aredescribed, for example, in Remingtons Pharmaceutical Sciences, MackPublishing Co. (A. R. Gennaro ed. 1985). In general, the type of carrieris selected based on the mode of administration. Pharmaceuticalcompositions may be formulated for any appropriate manner ofadministration, including, for example, topical, oral, nasal,intrathecal, rectal, vaginal, sublingual or parenteral administration,including subcutaneous, intravenous, intramuscular, intrasternal,intracavemous, intrameatal or intraurethral injection or infusion. Forparenteral administration, the carrier preferably comprises water,saline, alcohol, a fat, a wax or a buffer. For oral administration, anyof the above carriers or a solid carrier, such as mannitol, lactose,starch, magnesium stearate, sodium saccharine, talcum, cellulose,kaolin, glycerin, starch dextrins, sodium alginate,carboxymethylcellulose, ethyl cellulose, glucose, sucrose and/ormagnesium carbonate, may be employed.

[0102] A pharmaceutical composition (e.g., for oral administration ordelivery by injection) may be in the form of a liquid (e.g., an elixir,syrup, solution, emulsion or suspension). A liquid pharmaceuticalcomposition may include, for example, one or more of the following:sterile diluents such as water for injection, saline solution,preferably physiological saline, Ringer's solution, isotonic sodiumchloride, fixed oils such as synthetic mono or diglycerides which mayserve as the solvent or suspending medium, polyethylene glycols,glycerin, propylene glycol or other solvents; antibacterial agents suchas benzyl alcohol or methyl paraben; antioxidants such as ascorbic acidor sodium bisulfite; chelating agents such as ethylenediaminetetraaceticacid; buffers such as acetates, citrates or phosphates and agents forthe adjustment of tonicity such as sodium chloride or dextrose. Aparenteral preparation can be enclosed in ampoules, disposable syringesor multiple dose vials made of glass or plastic. The use ofphysiological saline is preferred, and an injectable pharmaceuticalcomposition is preferably sterile.

[0103] The compositions described herein may be formulated for sustainedrelease (i.e., a formulation such as a capsule or sponge that effects aslow release of compound following administration). Such compositionsmay generally be prepared using well known technology and administeredby, for example, oral, rectal or subcutaneous implantation, or byimplantation at the desired target site. Sustained-release formulationsmay contain an agent dispersed in a carrier matrix and/or containedwithin a reservoir surrounded by a rate controlling membrane. Carriersfor use within such formulations are biocompatible, and may also bebiodegradable; preferably the formulation provides a relatively constantlevel of active component release. The amount of active compoundcontained within a sustained release formulation depends upon the siteof implantation, the rate and expected duration of release and thenature of the condition to be treated or prevented.

[0104] Within a pharmaceutical composition, a therapeutic agentcomprising a polypeptide-directed siRNA polynucleotide as describedherein (or, e.g., a recombinant nucleic acid construct encoding a siRNApolynucleotide) may be linked to any of a variety of compounds. Forexample, such an agent may be linked to a targeting moiety (e.g., amonoclonal or polyclonal antibody, a protein or a liposome) thatfacilitates the delivery of the agent to the target site. As usedherein, a “targeting moiety” may be any substance (such as a compound orcell) that, when linked to an agent enhances the transport of the agentto a target cell or tissue, thereby increasing the local concentrationof the agent. Targeting moieties include antibodies or fragmentsthereof, receptors, ligands and other molecules that bind to cells of,or in the vicinity of, the target tissue. An antibody targeting agentmay be an intact (whole) molecule, a fragment thereof, or a functionalequivalent thereof. Examples of antibody fragments are F(ab′)₂, Fab′,Fab and F[v] fragments, which may be produced by conventional methods orby genetic or protein engineering. Linkage is generally covalent and maybe achieved by, for example, direct condensation or other reactions, orby way of bi- or multi-functional linkers. Targeting moieties may beselected based on the cell(s) or tissue(s) toward which the agent isexpected to exert a therapeutic benefit.

[0105] Pharmaceutical compositions may be administered in a mannerappropriate to the disease to be treated (or prevented). An appropriatedosage and a suitable duration and frequency of administration will bedetermined by such factors as the condition of the patient, the type andseverity of the patient's disease, the particular form of the activeingredient and the method of administration. In general, an appropriatedosage and treatment regimen provides the agent(s) in an amountsufficient to provide therapeutic and/or prophylactic benefit (e.g., animproved clinical outcome, such as more frequent complete or partialremissions, or longer disease-free and/or overall survival, or alessening of symptom severity). For prophylactic use, a dose should besufficient to prevent, delay the onset of or diminish the severity of adisease associated with cell proliferation.

[0106] Optimal dosages may generally be determined using experimentalmodels and/or clinical trials. In general, the amount of siRNApolynucleotide present in a dose, or produced in situ by DNA present ina dose (e.g., from a recombinant nucleic acid construct comprising asiRNA polynucleotide), ranges from about 0.01 μg to about 100 μg per kgof host, typically from about 0.1 μg to about 10 μg. The use of theminimum dosage that is sufficient to provide effective therapy isusually preferred. Patients may generally be monitored for therapeuticor prophylactic effectiveness using assays suitable for the conditionbeing treated or prevented, which will be familiar to those havingordinary skill in the art. Suitable dose sizes will vary with the sizeof the patient, but will typically range from about 10 mL to about 500mL for 10-60 kg animal.

[0107] The following Examples are offered by way of illustration and notby way of limitation.

EXAMPLE 1 Interference of TCPTP Expression by Specific siRNA

[0108] This Example describes the effect on expression of TCPTPexpression in cells transfected with sequence-specific siRNApolynucleotides.

[0109] The siRNA nucleotide sequences specific for each TCPTP werechosen by first scanning the open reading frame of the target cDNA for21-base sequences that were flanked on the 5′ end by two adenine bases(AA) and that had A+T/G+C ratios that were nearly 1:1. Twenty-one-basesequences with an A+T/G+C ratio greater than 2:1 or 1:2 were excluded.If no 21-base sequences were identified that met this criteria, thepolynucleotide sequence encoding the TCPTP was searched for a 21-basesequence having the bases CA at the 5′ end. The specificity of each21-mer was determined by performing a BLAST search of public databases.Sequences that contained at least 16 of 21 consecutive nucleotides with100% identity with a polynucleotide sequence other than the targetsequence were not used in the experiments.

[0110] Sense and antisense oligonucleotides for TCPTP analysis weresynthesized according to the standard protocol of the vendor (DharmaconResearch, Inc., Lafayette, Colo.). For some experiments described inthis and other examples, the vendor gel-purified the double-strandedsiRNA polynucleotide, which was then used. In the instances when thevendor did not prepare double-stranded siRNA, just before transfection,double-stranded siRNAs were prepared by annealing the sense andanti-sense oligonucleotides in annealing buffer (100 mM potassiumacetate, 30 mM HEPES-KOH, pH 7.4, 2 mM magnesium acetate) for 1 minuteat 90° C., followed by a 60-minute incubation at 37° C.

[0111] In each of the examples, each siRNA sequence represents the sensestrand of the siRNA polynucleotide and its corresponding sequenceidentifier. “Related sequence identifiers” referred to in the Examplesidentify sequences in the sequence listing that contain the samenucleotides at positions 1-19 of the siRNA sequence with and without twoadditional nucleotides (NN) at the 3′ end (which would correspond to atwo-nucleotide overhang in a double stranded polynucleotide), and thereverse complement of each. (It is noted that each 21-mer sequence thuscontains a dinucleotide “overhang” at the 3′ end, and that the inventionherein should be considered to include the 19-mer polynucleotidesequences beginning at the 5′ end therein as well as the 21-merpolynucleotide.) Unless otherwise stated, it is to be understood thatthe siRNA transfected into a cell is composed of the sense strand andits complementary antisense strand, which form a duplex siRNApolynucleotide.

[0112] Interference with Expression of Murine TCPTP by siRNA inCo-Transfection Assays

[0113] A co-transfection assay was performed in which 1BKO+HIR murinefibroblasts were co-transfected with an expression vector comprising apolynucleotide sequence (SEQ ID NO:______) encoding murine TCPTP (SEQ IDNO:______) and siRNA mTCPTP1.1 (5′-guugucaugcuaaaccgaact-3′ (SEQ IDNO:______)) (1 nM) or mTCPTP1.2 (5′-cagaacagagugaugguugag-3′ (SEQ IDNO:______)) (20 nM). The level of TCPTP expression was determined byimmunoblotting with an anti-human TCPTP antibody (Curt Diltz, CEPTYR,Inc.). A co-transfection assay was performed in which 1BKO+HIR murinefibroblasts. PTP1B KO mouse embryonic fibroblasts were prepared from13-day embryos from PTP1B knock out mice to establish the cell line,which was then transfected with human insulin receptor (1BKO+HIR) (HIR,Julie Moyers, Eli Lilly and Company, Indianapolis, Ind.)). Cells weretransfected with siRNAs or annealing buffer alone. Each siRNA wasdiluted in 250 μl O_(PTI)MEM® low serum medium (Gibco, Inc.) to a finalconcentration of 20 nM. In a separate tube, 10 μl of Lipofectamine® 2000(Invitrogen Life Technologies, Carlsbad, Calif.) was combined with 250μl O_(PTI)MEM®. Each solution was incubated for 7 minutes. The twosolutions were then mixed and incubated at room temperature for 22minutes. The final volume of the mixed solution was adjusted to 100 μland then the cells were added. The transfected cells were incubated22-24 hours at 37°.

[0114] Cell lysates were prepared by extracting the cells in ELISAextraction buffer (50 mM Tris-HCl, pH 7.5 (room temperature); 2 mM EDTA,pH 7-8; 1 mM phosphate (polyphosphate); 1 mM NaVO4 (monomeric), pH 10;0.1% Triton X-100; Protease Inhibitor Cocktail set III, (Calbiochem, SanDiego, Calif., catalog #539134)). The lysates were separated by SDS-PAGEgel and analyzed by immunoblot. The lysates were centrifuged andaliquots of supernatant (10 μl) from each transfected cell culturesample were combined with 10 μl of SDS-PAGE reducing sample buffer. Thesamples were heated at 95° C. for five minutes, and then applied to a14% Tris-glycine SDS-PAGE gel (NOVEX® from Invitrogen Life Technologies,Carlsbad, Calif.). After electrophoresis, the separated proteins wereelectrophoretically transferred from the gel onto an Immobilon-Ppolyvinylidene fluoride (PVDF) membrane (Millipore, Bedford, Mass.). ThePVDF membrane was blocked in 5% milk in TBST (20 mM Tris pH 7.5, 150 mMNaCl, 0.05% Tween-20); incubated with an anti-human TCPTP antibody (CurtDiltz, CEPTYR, Inc.) for 2-16 hours at room temperature; washed 3×10minutes with TBST; and then incubated with an appropriate horseradishperoxidase (HRP) conjugate IgG (1:10,000) (Amersham Biosciences,Piscataway, N.J.) for 30 minutes at room temperature. Binding wasdetected with the ECL chemiluminescent reagent used according to thevendor's instructions (Amersham Biosciences, Piscataway, N.J.).

[0115] The siRNA mTCPTP1.2 did not interfere with expression of murineTCPTP. Expression of murine TCPTP decreased more than 95% in cellstransfected with siRNA, mTCPTP1.1.

[0116] Interference with Expression of Human TCPTP by siRNA inCo-Transfection Assays

[0117] Co-transfection assays were performed essentially as describedabove to determine siRNA inhibition of human TCPTP expression. Arecombinant expression construct was prepared that encodes wild-typehuman TC45. The following oligonucleotide primers were used for thewild-type construct. The sequences of the BamHI and EcoRi restrictionsites are underlined. Human TC45 sense (TC45 5′BamHI)5′-GGGGGGATCCATGCCCACCACCATCGAGCGGGAGTT-3′ (SEQ ID NO_) Human TC45antisense (TC45 3′EcoRI)5′-GGGGAATTCTTAGGTGTCTGTCAATCTTGGCCTTTTTCTTTTTCGTTCA-3′ (SEQ ID NO:_)

[0118] Vector pCMVTag2B (Stratagene, La Jolla, Calif.) was digested withrestriction endonuclease BamHI (New England Biolabs, Beverly, Mass.) for3 hours at 37° C. The digested vector was then incubated with Klenowpolymerase (New England Biolabs) for 15 minutes at 25° C. to fill in therecessed 3′ termini, followed by an incubation of 30 minutes at 37° C.with calf intestinal phosphatase (New England Biolabs). The GATEWAY™Reading Frame Cassette B (Invitrogen Life Technologies) was insertedinto the pCMVTag2B vector by ligation with T4 DNA ligase (InvitrogenLife Technologies) overnight at 16° C. according to the supplier'sinstructions. DB3.1™ competent E. coli cells were transformed with theligated vector (GWpCMVTag2) and DNA was isolated by standard molecularbiology methods.

[0119] Vectors for expression of TC45 wild type were prepared asfollows: The TC45 construct was subcloned into a GATEWAY™ entry vectorpENTR3C™ (Invitrogen Life Technologies) by digesting 10 μl of the TC45cDNA with 1 μl of BamHI (New England Biolabs), 1 μl of EcoRI (NewEngland Biolabs), 3 μl 10×EcoRI buffer (New England Biolabs), 3 μl10×BSA (New England Biolabs), and 12 μl distilled water for 3 hours at37° C. Two microliters of the pENTR3C™ vector was digested with 0.5 μlof BamHI (New England Biolabs), 0.5 μl of EcoRI (New England Biolabs), 2μl 10×EcoRI buffer (New England Biolabs), 2 μl 10×BSA (New EnglandBiolabs), and 13 μl distilled water for 3 hours at 37° C., followed byan incubation of 30 minutes at 37° C. with calf intestinal phosphatase(New England Biolabs). Digested DNA was run on a 1% agarose gel,digested bands were excised and gel purified using a QIAGEN GelExtraction kit (QIAGEN, Inc.). Four microliters of the TC45 cDNA wasligated into 2 μl of the pENTR3C™ vector overnight at 16° C. with 1 μl10×Ligation Buffer (Invitrogen Life Technologies), 1 μl T4 DNA Ligase(4U/μl) (Invitrogen Life Technologies), and 2 μl distilled water. Theconstruct was transformed into LIBRARY EFFICIENCY® DH5α™ cells. TheFLAG® epitope-tagged TC45 construct was prepared by cloning the pENTR3C™TC45 WT construct into the GWpCMVTag2 vector. The pENTR3C™ constructcontaining the TC45 polynucleotide was linearized by digesting theconstruct with Pvu I (New England Biolabs)) at 37° C. for 2 hours. TheDNA was purified using a QIAGEN PCR Purification kit (QIAGEN, Inc.). Twomicroliters (150 ng/μl) of the GWpCMVTag2 vector were combined in aGATEWAY™ LR reaction with 3 μl linearized pENTR3C™ TC45 WT, 5 μl TEbuffer, 4 μl Clonase™ Enzyme, and 4 μl LR reaction buffer (InvitrogenLife Technologies) overnight at room temperature. After addition ofProteinase K (Invitrogen Life Technologies) to the reaction for 10minutes, LIBRARY EFFICIENCY® DH5α™ cells were transformed with theexpression construct.

[0120] Cells (1BKO+HIR murine embryo fibroblasts) were co-transfected(see method above) with an expression vector containing a nucleotidesequence encoding human TCPTP (SEQ ID NO:______) and siRNAs, hTCPTP1.4(5′-guugucaugcugaaccgcatt-3′ (SEQ ID NO:______)) (20 nM); hTCPTP1.5(5′-gcccauaugaucacagucgtg-3′ (SEQ ID NO:______)) (10 nM); hTCPTP1.6(5′-ucgguuaaaugugcacaguac-3′ (SEQ ID NO:______)) (10 nM); or hTCPTP1.7(5′-ugacuauccucauagaguggg-3′ (SEQ ID NO:______)) (20 nM). Additionalhuman TCPTP specific siRNA polynucleotides were prepared; the sequencesof each are as follows: hTCPTP1.1 (5′-agugagagaaucuggcucctt-3′ (SEQ IDNO:______)); hTCPTP1.2 (5′-ggaagacuuaucuccugcctt-3′ (SEQ ID NO:______));and hTCPTP1.3 (5′-ggugaccgauguacaggactt-3′ (SEQ ID NO:______)). Thelevel of TCPTP expression was determined by immunoblotting with ananti-human TCPTP antibody. The level of expression of human TCPTP wasnot affected by siRNA hTCPTP1.7. Expression levels decreased more than95% in the cells co-transfected with hTCPTP1.4; 80% in cellsco-transfected with hTCPTP1.5; and greater than 90% in cells transfectedwith hTCPTP1.6.

[0121] Interference of Endogenous Expression of Human TCPTP by siRNA

[0122] 293-HEK HIR cells were transfected with either hTCPTP1.4 (SEQ IDNO:______) or rPTP1B1.2, a rat PTP1B sequence specific siRNA(5′-cggaugguggguggagguctt-3′ (SEQ ID NO:______), which was included as anonspecific siRNA control, at concentrations of 2, 5, 10, 20, and 50 nM.Cells were plated one day prior to transfection in either a 24-well or6-well format. The siRNA polynucleotide to be added to each well wasdiluted into 50 μl O_(PTI)MEM® to the final concentrations listed above.In a separate vessel, 3 μl Oligofectamine™ (Invitrogen LifeTechnologies) was diluted into 12 μl O_(PTI)MEM®. Each solution wasincubated for 7 minutes at room temperature, after which the solutionswere mixed and incubated for 22 minutes. The final volume of the mixedsolution was brought to 100 μl with O_(PTI)MEM®. Cells were transfectedwith the specific siRNA polynucleotides, non-specific siRNApolynucleotides, and annealing buffer alone, and incubated at 37° for48-72 hours. Endogenous expression of human TCPTP in the cellstransfected with sequence specific hTCPTP1.4 decreased 90%.

EXAMPLE 2 Regulatory Role of TCPTP in Insulin Signaling

[0123] The protein tyrosine phosphatase TC-PTP exists in twoalternatively spliced forms, TC45 and TC48, that share the samecatalytic domain but differ at their extreme carboxy-termini (Mosingeret al., Proc. Natl. Acad. Sci. USA 89:499-503 (1992)). Insulin-inducedoxidation and inactivation of TC45 suggested that it functions as anegative regulator of insulin signaling (see U.S. Ser. No. 10/366,547).This Example examines the regulatory role of TC45 in insulin signalingby inhibiting expression of the PTP by RNAi.

[0124] The specific siRNA duplexes were designed by first scanningthrough the open reading frame of TC45 mRNA and selecting sequences of5′AA(N₁₉)3′ (N=any nucleotide) for further characterization. Thefollowing 2 oligonucleotides were chosen: 5′-AACAGAUACAGAGAUGUAAGC-3′(TCPTP1) (SEQ ID NO:______) and 5′-AAGCCCA UAUGAUC ACAGUCG -3′ (TCPTP2)(SEQ ID NO:______). These sequences were submitted to a BLAST searchagainst human, rat, and mouse genome databases to ensure specificity forTC-PTP. The 21-nt siRNA duplexes were obtained in a deprotected anddesalted form (Dharmacon Research). Rat-1 fibroblasts (Fischer ratfibroblast 3T3 like cell line) and HepG2 (human hepatocellularcarcinoma) cells (American Type Culture Collection (ATCC), Manasass,Va.) were transfected with each siRNA at 100 nM. Both siRNAoligonucleotides suppressed expression of endogenous TC45 in thetransfected HepG2 cells and Rat-1 fibroblasts, with TCPTP1 being moreefficient.

[0125] Rat-1 (fibroblasts) and HepG2 (human hepatocellular carcinoma)cells were routinely maintained in DMEM supplemented with 10% FBS, 1%glutamine, 100 U/ml penicillin and 100 μg/ml streptomycin. Forstimulation with insulin, cells were plated in media containing 10% FBSfor 48 hours, then serum-starved for 16 hours before treatment. Fortransient transfection, cells were plated in DMEM supplemented with 10%FBS for 16 hours, then in OptiMEM (Invitrogen) without serum, afterwhich the plasmid (5 μg/dish for Rat-1, 30 μg/dish for HepG2) wasintroduced by LipofectAMINE and PLUS reagents (Invitrogen), according tothe manufacture's recommendations. The transfection efficiency wasroutinely 40%. For RNAi experiments, cells were plated as above and theTCPTP siRNA duplexes were introduced by Oligofectamine (Invitrogen)according to the guidelines provided by Dharmacon Research Inc.

[0126] The potential regulatory role of TC45 in insulin signaling wasinvestigated by examining the phosphorylation status of PKB/Akt, whichis a critical effector in the PI3 kinase pathway that mediates variousintracellular responses to insulin, following ablation of the PTP byRNAi. The human hepatoma cell line HepG2 has been used extensively as amodel to study insulin signaling (see Huang et al., J. Biol. Chem.277:18151-60 (2002); Haj et al., Science 295 1708-11 (2002)).Serum-deprived Rat-1 and HepG2 cells were exposed to 10 or 50 nM insulinfor 5 min and lysed. The insulin receptor (IR) was immunoprecipitatedfrom 500 μg of cell lysate with anti-IR-β antibody 29B4 (Santa CruzBiotechnology), then immunoblotted with anti-phosphotyrosine,anti-pYpY^(1162/1162)-IR-β (Biosource International, Camarillo, Calif.)and anti-IR-β (C-19) (Santa Cruz Biotechnology) antibodies. HepG2 cellsexpressed higher levels of IR-β than Rat-1 cells as shown in FIG. 1A anddisplayed a robust response to insulin stimulation, as shown by theoverall tyrosine phosphorylation level of IR-β and autophosphorylationof the activation loop tyrosines 1162 and 1163 (see FIG. 1A).

[0127] For the RNAi experiment, HepG2 cells were untransfected (control)or transfected (+siRNA) with 100 nM siRNA TCPTP1 oligonucleotide. Twodays after transfection, cells were serum-starved for 16 hours and thenstimulated with 10 nM insulin for 0, 1, 2, 5, 10, and 20 minutes. Totallysates (30 μg) were immunoblotted with anti-phospho-PKB/Akt (CellSignaling Technology, Beverly, Mass.); anti-PKB/Akt (Cell SignalingTechnology); anti-TC45 (1910H (Lorenzen et al., J. Cell. Biol.131:631-43 (1995))); and anti-PTP1B (FG6 (LaMontagne et al., Mol. Cell.Biol. 18:2965-75 (1998))) antibodies. The results presented in FIG. 1Bindicate that depletion of TC45 enhanced both the intensity and durationof the signaling response. FIG. 1C illustrates a densitometric analysisof the gel image to show the ratio of phosphorylated PKB/Akt relative tototal PKB/Akt. Similar results were observed in three independentexperiments.

[0128] The role of TC45 in insulin signaling was further investigated bypreparing a TC45 substrate trapping mutant. Substitution of an alanineresidue for the invariant aspartate, which functions as a general acidin catalysis, into the vector expressing TC45 and into a vectorexpressing PTP1B was performed by standard site-directed mutagenesisprotocols. HepG2 cells overexpressing wild type (WT) or trapping mutant(DA) forms of PTP1B and TC45 were either left untreated (−INS) orstimulated with 10 nM insulin for 5 min (+INS), then lysed in trappingbuffer (20 mM Tris (pH 7.4), 1% NP-40, 150 mM NaCl, 10% glycerol, 10 mMIAA and 25 μg/ml each of aprotinin and leupeptin). Aliquots (1 mg) ofcell lysate were incubated with anti-PTP1B antibody (FG6) or anti-TC45antibody (CF4). The immunocomplexes were washed with lysis buffer,subjected to SDS-PAGE then immunoblotted with anti-IR-β (C-19) antibody.An aliquot of lysate (30 μg) was immunoblotted with anti-PTP1B antibody(FG6) or anti-TC-PTP antibody (CF4) to verify PTP expression. The dataare shown in FIG. 2A and are representative of three independentexperiments. These data suggest that TC45 recognizes IR-β as asubstrate.

[0129] Serum starved, untransfected (control) or TC45 siRNA (100 nM)transfected (+siRNA) HepG2 cells were stimulated with 10 nM insulin for0, 1, 2, 5, 10, and 20 minutes. The insulin receptor wasimmunoprecipitated from 750 μg of cell lysate with anti-IR-β antibody29B4 and immunoblotted with anti-phosphotyrosine (G104), anti-pY⁹⁷²-IR-β(Biosource), anti-pYpY^(1162/1163)-IR-β, and anti-IR-β (C-19) antibodiesas shown in FIG. 2B. FIG. 2C illustrates densitometric analyses of thegel image to show the ratio of phosphorylated IR-β relative to totalIR-β for total phosphotyrosine (upper panel), phosphorylation of Tyr 972(middle panel), and phosphorylation of the activation loop tyrosines1162 and 1163 (lower panel). Similar results were observed in twoindependent experiments.

EXAMPLE 3 Effect of siRNAs Specific for TCPTP on Insulin ReceptorTyrosine Phosphorylation

[0130] This example illustrates the effect of RNAi on the function ofcomponents in a cell signaling pathway.

[0131] The effect of human TCPTP siRNA on the level of phosphorylationof IR-β was evaluated by ELISA. 292-HEK HIR cells were transfected with0, 0.5, 3, or 10 nM siRNAs. The siRNA polynucleotides transfected intothe cells included mPTP1B1.1 (SEQ ID NO:______) and hTCPTP1.4 (SEQ IDNO:______). Seventy-two hours after transfection, cells were exposed toinsulin for 7 minutes at concentrations of 0, 5, 10, 20, 50, and 100 nM.Cell lysates were prepared as described in Example 1, and total cellprotein was quantified by the Bio-Rad Protein Assay performed accordingto the manufacturer's instructions (Bio-Rad, Hercules, Calif.).

[0132] An ELISA was performed as follows. Dynex Immulon HB4X plates werecoated with anti-insulin receptor antibody Ab-1 (1 mg/ml; NeoMarkers,Inc., Fremont, Calif.) that was diluted 1:1000 in CMF (calcium magnesiumfree)-PBS containing 5 μg/ml fatty acid free BSA (faf-BSA). The plateswere incubated at 4° C. for at least four hours. The antibody solutionwas removed by aspiration, followed by the addition of 300 μl of 3%faf-BSA+CMF-PBS. The plates were incubated for 1 hr with agitation on avortex platform shaker (setting #5) at room temperature. Afteraspirating the 3% faf-BSA+CMF-PBS solution, approximately 10-20 μg oflysate were added to the wells and incubated at room temperature for onehour. Plates were washed three times with TBST (20 mM Tris, —HCl, pH 7.5150 mM NaCl; 0.05% Tween 20). An anti-insulin receptor phosphotyrosinespecific antibody (pTyr 1162/63, Biosource International, Camarillo,Calif., Catalog #44-804) was diluted 1:2000 in TBST and added to theplates for one hour at room temperature. The plates were washed threetimes with TBST. HRP-conjugated anti-rabbit antibody (AmershamBiosciences, catalog #NA934V) (1:2000 in TBST) was then added to thewells and incubated at room temperature for one hour. The plates werewashed three times with TBST and once with deionized, sterile water. TMBsolution (Sigma Aldrich) (100 μl per well) was added and developed untila modest color change (10-30 minutes depending on cell type and insulinresponse). The reaction was stopped with 100 μl of 1.8 N H₂SO₄ and thenmixed. The optical density of each well was measured at 450 nM in aSpectramax plate reader (Molecular Devices Corp., Sunnyvale, Calif.). Asshown in FIG. 3 increased phosphorylation of the insulin receptor wasobserved in cells transfected with hTCPTP1.4.

Additional References

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[0135] Brummelkamp et al., Science 296:550-53 (2002)

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[0158] EP1 152 056

[0159] U.S. Pat. No. 2001/0029617

[0160] U.S. Pat. No. 2002/0007051

[0161] U.S. Pat. No. 6,326,193

[0162] U.S. Pat. No. 6,342,595

[0163] U.S. Pat. No. 6,506,559

[0164] WO 01/29058

[0165] WO 01/34815

[0166] WO 01/42443

[0167] WO 01/68836

[0168] WO 01/75164

[0169] WO 01/92513

[0170] WO 01/96584

[0171] WO 99/32619

[0172] From the foregoing, it will be appreciated that, althoughspecific embodiments of the invention have been described herein for thepurpose of illustration, various modifications may be made withoutdeviating from the spirit and scope of the invention. Accordingly, thepresent invention is not limited except as by the appended claims.

1 76 1 7 PRT Unknown Unique signature sequence motif found in conserveddomain of the PTP family of enzymes 1 Cys Xaa Xaa Xaa Xaa Xaa Arg 1 5 211 PRT unknown 11 amino acid conserved sequence containing the signaturesequence motif found in a majority of PTPs 2 Xaa His Cys Xaa Ala Gly XaaXaa Arg Xaa Gly 1 5 10 3 2287 DNA Homo sapiens 3 ggggggcctg agcctctccgccggcgcagg ctctgctcgc gccagctcgc tcccgcagcc 60 atgcccacca ccatcgagcgggagttcgaa gagttggata ctcagcgtcg ctggcagccg 120 ctgtacttgg aaattcgaaatgagtcccat gactatcctc atagagtggc caagtttcca 180 gaaaacagaa atcgaaacagatacagagat gtaagcccat atgatcacag tcgtgttaaa 240 ctgcaaaatg ctgagaatgattatattaat gccagtttag ttgacataga agaggcacaa 300 aggagttaca tcttaacacagggtccactt cctaacacat gctgccattt ctggcttatg 360 gtttggcagc agaagaccaaagcagttgtc atgctgaacc gcattgtgga gaaagaatcg 420 gttaaatgtg cacagtactggccaacagat gaccaagaga tgctgtttaa agaaacagga 480 ttcagtgtga agctcttgtcagaagatgtg aagtcgtatt atacagtaca tctactacaa 540 ttagaaaata tcaatagtggtgaaaccaga acaatatctc actttcatta tactacctgg 600 ccagattttg gagtccctgaatcaccagct tcatttctca atttcttgtt taaagtgaga 660 gaatctggct ccttgaaccctgaccatggg cctgcggtga tccactgtag tgcaggcatt 720 gggcgctctg gcaccttctctctggtagac acttgtcttg ttttgatgga aaaaggagat 780 gatattaaca taaaacaagtgttactgaac atgagaaaat accgaatggg tcttattcag 840 accccagatc aactgagattctcatacatg gctataatag aaggagcaaa atgtataaag 900 ggagattcta gtatacagaaacgatggaaa gaactttcta aggaagactt atctcctgcc 960 tttgatcatt caccaaacaaaataatgact gaaaaataca atgggaacag aataggtcta 1020 gaagaagaaa aactgacaggtgaccgatgt acaggacttt cctctaaaat gcaagataca 1080 atggaggaga acagtgagagtgctctacgg aaacgtattc gagaggacag aaaggccacc 1140 acagctcaga aggtgcagcagatgaaacag aggctaaatg agaatgaacg aaaaagaaaa 1200 aggtggttat attggcaacctattctcact aagatggggt ttatgtcagt cattttggtt 1260 ggcgcttttg ttggctggagactgtttttt cagcaaaatg ccctataaac aattaatttt 1320 gcccagcaag cttctgcactagtaactgac agtgctacat taatcatagg ggtttgtctg 1380 cagcaaacgc ctcatatcccaaaaacggtg cagtagaata gacatcaacc agataagtga 1440 tatttacagt cacaagcccaacatctcagg actcttgact gcaggttcct ctgaacccca 1500 aactgtaaat ggctgtctaaaataaagaca ttcatgtttg ttaaaaactg gtaaattttg 1560 caactgtatt catacatgtcaaacacagta tttcacctga ccaacattga gatatccttt 1620 atcacaggat ttgtttttggaggctatctg gattttaacc tgcacttgat ataagcaata 1680 aatattgtgg ttttatctacgttattggaa agaaaatgac atttaaataa tgtgtgtaat 1740 gtataatgta ctattgacatgggcatcaac acttttattc ttaagcattt cagggtaaat 1800 atattttata agtatctatttaatcttttg tagttaactg tactttttaa gagctcaatt 1860 tgaaaaatct gttactaaaaaaaaaaattg tatgtcgatt gaattgtact ggatacattt 1920 tccatttttc taaaaagaagtttgatatga gcagttagaa gttggaataa gcaatttcta 1980 ctatatattg catttcttttatgttttaca gttttcccca ttttaaaaag aaaagcaaac 2040 aaagaaacaa aagtttttcctaaaaatatc tttgaaggaa aattctcctt actgggatag 2100 tcaggtaaac agttggtcaagactttgtaa agaaattggt ttctgtaaat cccattattg 2160 atatgtttat ttttcatgaaaatttcaatg tagttggggt agattatgat ttaggaagca 2220 aaagtaagaa gcagcattttatgattcata atttcagttt actagactga agttttgaag 2280 taaaccc 2287 4 415 PRTHomo sapiens 4 Met Pro Thr Thr Ile Glu Arg Glu Phe Glu Glu Leu Asp ThrGln Arg 1 5 10 15 Arg Trp Gln Pro Leu Tyr Leu Glu Ile Arg Asn Glu SerHis Asp Tyr 20 25 30 Pro His Arg Val Ala Lys Phe Pro Glu Asn Arg Asn ArgAsn Arg Tyr 35 40 45 Arg Asp Val Ser Pro Tyr Asp His Ser Arg Val Lys LeuGln Asn Ala 50 55 60 Glu Asn Asp Tyr Ile Asn Ala Ser Leu Val Asp Ile GluGlu Ala Gln 65 70 75 80 Arg Ser Tyr Ile Leu Thr Gln Gly Pro Leu Pro AsnThr Cys Cys His 85 90 95 Phe Trp Leu Met Val Trp Gln Gln Lys Thr Lys AlaVal Val Met Leu 100 105 110 Asn Arg Ile Val Glu Lys Glu Ser Val Lys CysAla Gln Tyr Trp Pro 115 120 125 Thr Asp Asp Gln Glu Met Leu Phe Lys GluThr Gly Phe Ser Val Lys 130 135 140 Leu Leu Ser Glu Asp Val Lys Ser TyrTyr Thr Val His Leu Leu Gln 145 150 155 160 Leu Glu Asn Ile Asn Ser GlyGlu Thr Arg Thr Ile Ser His Phe His 165 170 175 Tyr Thr Thr Trp Pro AspPhe Gly Val Pro Glu Ser Pro Ala Ser Phe 180 185 190 Leu Asn Phe Leu PheLys Val Arg Glu Ser Gly Ser Leu Asn Pro Asp 195 200 205 His Gly Pro AlaVal Ile His Cys Ser Ala Gly Ile Gly Arg Ser Gly 210 215 220 Thr Phe SerLeu Val Asp Thr Cys Leu Val Leu Met Glu Lys Gly Asp 225 230 235 240 AspIle Asn Ile Lys Gln Val Leu Leu Asn Met Arg Lys Tyr Arg Met 245 250 255Gly Leu Ile Gln Thr Pro Asp Gln Leu Arg Phe Ser Tyr Met Ala Ile 260 265270 Ile Glu Gly Ala Lys Cys Ile Lys Gly Asp Ser Ser Ile Gln Lys Arg 275280 285 Trp Lys Glu Leu Ser Lys Glu Asp Leu Ser Pro Ala Phe Asp His Ser290 295 300 Pro Asn Lys Ile Met Thr Glu Lys Tyr Asn Gly Asn Arg Ile GlyLeu 305 310 315 320 Glu Glu Glu Lys Leu Thr Gly Asp Arg Cys Thr Gly LeuSer Ser Lys 325 330 335 Met Gln Asp Thr Met Glu Glu Asn Ser Glu Ser AlaLeu Arg Lys Arg 340 345 350 Ile Arg Glu Asp Arg Lys Ala Thr Thr Ala GlnLys Val Gln Gln Met 355 360 365 Lys Gln Arg Leu Asn Glu Asn Glu Arg LysArg Lys Arg Trp Leu Tyr 370 375 380 Trp Gln Pro Ile Leu Thr Lys Met GlyPhe Met Ser Val Ile Leu Val 385 390 395 400 Gly Ala Phe Val Gly Trp ArgLeu Phe Phe Gln Gln Asn Ala Leu 405 410 415 5 462 DNA Homo sapiens 5cggaaacgta ttcgagagga cagaaaggcc accacagctc agaaggtgca gcagatgaaa 60cagaggctaa atgagaatga acgaaaaaga aaaaggccaa gattgacaga cacctaatat 120tcatgactta agaatattct gcagctataa attttgaacc attgatgtgc aaagcaagac 180ctgaagccca ctccggaaac taaagtgagg ctcgctaacc ctctagattg cctcacagtt 240gtttgtttac aaagtaaact ttacatccag gggatgaaga gcacccacca gcagaagact 300ttgcagaacc tttaattgga tgtgttaagt gtttttaatg agtgtatgaa atgtagaaag 360atgtacaaga aataaattag gagagattac tttgtattgt actgccattc ctactgtatt 420tttatacttt ttggcagcat taaatatttt tgttaaatag tc 462 6 462 DNA Homosapiens 6 cggaaacgta ttcgagagga cagaaaggcc accacagctc agaaggtgcagcagatgaaa 60 cagaggctaa atgagaatga acgaaaaaga aaaaggccaa gattgacagacacctaatat 120 tcatgactta agaatattct gcagctataa attttgaacc attgatgtgcaaagcaagac 180 ctgaagccca ctccggaaac taaagtgagg ctcgctaacc ctctagattgcctcacagtt 240 gtttgtttac aaagtaaact ttacatccag gggatgaaga gcacccaccagcagaagact 300 ttgcagaacc tttaattgga tgtgttaagt gtttttaatg agtgtatgaaatgtagaaag 360 atgtacaaga aataaattag gagagattac tttgtattgt actgccattcctactgtatt 420 tttatacttt ttggcagcat taaatatttt tgttaaatag tc 462 7 1555DNA Mus musculus 7 tctccccgga tagagcgggg cccgagcctg tccgctgtggtagttccgct cggctgcccc 60 gccgccatgt cggcaaccat cgagcgggag ttcgaggaactggatgctca gtgtcgctgg 120 cagccgttat acttggaaat tcgaaatgaa tcccatgactatcctcatag agtggccaag 180 tttccagaaa acagaaaccg aaacagatac agagatgtaagcccatatga tcacagtcgt 240 gttaaactgc aaagtactga aaatgattat attaatgccagcttagttga catagaagag 300 gcacaaagaa gttacatctt aacacagggc ccacttccgaacacatgctg ccatttctgg 360 ctcatggtgt ggcagcaaaa gaccaaagca gttgtcatgctaaaccgaac tgtagaaaaa 420 gaatcggtta aatgtgcaca gtactggcca acggatgacagagaaatggt gtttaaggaa 480 acgggattca gtgtgaagct cttatctgaa gatgtaaaatcatattatac agtacatcta 540 ctacagttag aaaatatcaa tactggtgaa acgagaaccatatctcactt ccattatacc 600 acctggccag attttggggt tccagagtca ccagcttcatttctaaactt cttgtttaaa 660 gttagagaat ctggttgttt gacccctgac catggacctgcagtgatcca ttgcagtgcg 720 ggcatcgggc gctctggcac cttctctctt gtagatacctgtcttgttct gatggaaaaa 780 ggagaggatg ttaatgtgaa acaattatta ctgaatatgagaaagtatcg aatgggactt 840 attcagacac cggaccaact cagattctcc tacatggccataatagaagg agcaaagtac 900 acaaaaggag attcaaatat acagaaacgg tggaaagaactttctaaaga agatttatct 960 cctatttgtg atcattcaca gaacagagtg atggttgagaagtacaatgg gaagagaata 1020 ggttcagaag atgaaaagtt aacagggctt ccttctaaggtgcaggatac tgtggaggag 1080 agcagtgaga gcattctacg gaaacgtatt cgagaggatagaaaggctac gacggctcag 1140 aaggtgcagc agatgaaaca gaggctaaat gaaactgaacgaaaaagaaa aaggccaaga 1200 ttgacagaca cctaaatgtt catgacttga gactattctgcagctataaa atttgaacct 1260 ttgatgtgca aagcaagacc tgaagcccac tccggaaactaaagtgaggc ttgctaaccc 1320 tgtagattgc ctcacaagtt gtctgtttac aaagtaagctttccatccag gggatgaaga 1380 acgccaccag cagaagactt gcaaaccctt taatttgatgtattgttttt taacatgtgt 1440 atgaaatgta gaaagatgta aaggaaataa attaggagcgactactttgt attgtactgc 1500 cattcctaat gtatttttat actttttggc agcattaaatatttttatta aatag 1555 8 382 PRT Mus musculus 8 Met Ser Ala Thr Ile GluArg Glu Phe Glu Glu Leu Asp Ala Gln Cys 1 5 10 15 Arg Trp Gln Pro LeuTyr Leu Glu Ile Arg Asn Glu Ser His Asp Tyr 20 25 30 Pro His Arg Val AlaLys Phe Pro Glu Asn Arg Asn Arg Asn Arg Tyr 35 40 45 Arg Asp Val Ser ProTyr Asp His Ser Arg Val Lys Leu Gln Ser Thr 50 55 60 Glu Asn Asp Tyr IleAsn Ala Ser Leu Val Asp Ile Glu Glu Ala Gln 65 70 75 80 Arg Ser Tyr IleLeu Thr Gln Gly Pro Leu Pro Asn Thr Cys Cys His 85 90 95 Phe Trp Leu MetVal Trp Gln Gln Lys Thr Lys Ala Val Val Met Leu 100 105 110 Asn Arg ThrVal Glu Lys Glu Ser Val Lys Cys Ala Gln Tyr Trp Pro 115 120 125 Thr AspAsp Arg Glu Met Val Phe Lys Glu Thr Gly Phe Ser Val Lys 130 135 140 LeuLeu Ser Glu Asp Val Lys Ser Tyr Tyr Thr Val His Leu Leu Gln 145 150 155160 Leu Glu Asn Ile Asn Thr Gly Glu Thr Arg Thr Ile Ser His Phe His 165170 175 Tyr Thr Thr Trp Pro Asp Phe Gly Val Pro Glu Ser Pro Ala Ser Phe180 185 190 Leu Asn Phe Leu Phe Lys Val Arg Glu Ser Gly Cys Leu Thr ProAsp 195 200 205 His Gly Pro Ala Val Ile His Cys Ser Ala Gly Ile Gly ArgSer Gly 210 215 220 Thr Phe Ser Leu Val Asp Thr Cys Leu Val Leu Met GluLys Gly Glu 225 230 235 240 Asp Val Asn Val Lys Gln Leu Leu Leu Asn MetArg Lys Tyr Arg Met 245 250 255 Gly Leu Ile Gln Thr Pro Asp Gln Leu ArgPhe Ser Tyr Met Ala Ile 260 265 270 Ile Glu Gly Ala Lys Tyr Thr Lys GlyAsp Ser Asn Ile Gln Lys Arg 275 280 285 Trp Lys Glu Leu Ser Lys Glu AspLeu Ser Pro Ile Cys Asp His Ser 290 295 300 Gln Asn Arg Val Met Val GluLys Tyr Asn Gly Lys Arg Ile Gly Ser 305 310 315 320 Glu Asp Glu Lys LeuThr Gly Leu Pro Ser Lys Val Gln Asp Thr Val 325 330 335 Glu Glu Ser SerGlu Ser Ile Leu Arg Lys Arg Ile Arg Glu Asp Arg 340 345 350 Lys Ala ThrThr Ala Gln Lys Val Gln Gln Met Lys Gln Arg Leu Asn 355 360 365 Glu ThrGlu Arg Lys Arg Lys Arg Pro Arg Leu Thr Asp Thr 370 375 380 9 1494 DNARattus norvegicus 9 ttccgctcgc gctcccccgc cgccatgtcg gctaccatcgagcgggagtt cgaggaactg 60 gatgctcagt gtcgctggca gccgttatac ttggaaattcgaaatgaatc ccatgactat 120 cctcatagag tggccaagtt tccagaaaac agaaatcgaaacagatacag agatgtaagc 180 ccatatgatc acagtcgtgt taaactgcag agtgctgaaaatgattatat taatgccagc 240 ttagttgaca tagaagaggc acaaagaagt tacatcttaacacagggccc acttcctaac 300 acgtgctgcc atttctggct catggtgtgg cagcaaaagaccagagcagt tgtcatgcta 360 aaccgaactg tagagaaaga atcggttaaa tgtgcacagtactggccaac ggatgaccga 420 gagatggtgt ttaaggaaac aggattcagc gtgaagctcttatctgaaga tgtgaaatca 480 tattatacag tacatctact acagttagaa aatatcaatagtggtgaaac cagaaccata 540 tctcactttc attataccac ctggccagat tttggcgttccggagtcacc agcttcattc 600 ctaaatttct tgtttaaagt tagagaatct ggttctttgaaccctgacca tgggcctgca 660 gtgatccatt gcagtgcagg catcgggcgt tctggcaccttctctcttgt agatacctgt 720 ctcgttctga tggagaaagg agaggatgtt aatgtgaaacaaatattact gagtatgaga 780 aagtatcgaa tgggactcat tcagactccg gaccagctcagattctccta catggccata 840 atagaaggag caaagtatac aaaaggagat tcaaatatacagaacagaac aatgactgag 900 aagtacaacg ggaagagaat agggtcagaa gatgaaaagttaacaggact ttcttctaag 960 gttccagata ctgtggaaga gagcagtgag agtattctccggaaacgcat tcgagaggat 1020 agaaaggcta caaccgctca gaaggtgcag cagatgagacagaggctaaa tgaaactgaa 1080 cggaaaagga aaaggccaag attgacagac acctaaatgttcatgacttg agactattct 1140 gcagctataa attttgaacc tttgatgtgc aaagcaagacctgaagccca ctccggaaac 1200 taaagtgagg cttgctaacc ctgtagattg cctcacaagttgtctgttta caaagtaagc 1260 tttacatcca ggggatgaag aacgccacca gcagaagacttgcaaaccct ttaatttgac 1320 gtattgtttt ttaacatgtg tatgaattgt agaaagatgtaaagaaaata aaattaggag 1380 agactacttt gtattgtact gccattccta atgtatttttatactttttg gcagcattaa 1440 atatttttat taaatagaca aaaaaaaaaa aaaaaaaaaaaaaaaaaaaa aaaa 1494 10 363 PRT Rattus norvegicus 10 Met Ser Ala Thr IleGlu Arg Glu Phe Glu Glu Leu Asp Ala Gln Cys 1 5 10 15 Arg Trp Gln ProLeu Tyr Leu Glu Ile Arg Asn Glu Ser His Asp Tyr 20 25 30 Pro His Arg ValAla Lys Phe Pro Glu Asn Arg Asn Arg Asn Arg Tyr 35 40 45 Arg Asp Val SerPro Tyr Asp His Ser Arg Val Lys Leu Gln Ser Ala 50 55 60 Glu Asn Asp TyrIle Asn Ala Ser Leu Val Asp Ile Glu Glu Ala Gln 65 70 75 80 Arg Ser TyrIle Leu Thr Gln Gly Pro Leu Pro Asn Thr Cys Cys His 85 90 95 Phe Trp LeuMet Val Trp Gln Gln Lys Thr Arg Ala Val Val Met Leu 100 105 110 Asn ArgThr Val Glu Lys Glu Ser Val Lys Cys Ala Gln Tyr Trp Pro 115 120 125 ThrAsp Asp Arg Glu Met Val Phe Lys Glu Thr Gly Phe Ser Val Lys 130 135 140Leu Leu Ser Glu Asp Val Lys Ser Tyr Tyr Thr Val His Leu Leu Gln 145 150155 160 Leu Glu Asn Ile Asn Ser Gly Glu Thr Arg Thr Ile Ser His Phe His165 170 175 Tyr Thr Thr Trp Pro Asp Phe Gly Val Pro Glu Ser Pro Ala SerPhe 180 185 190 Leu Asn Phe Leu Phe Lys Val Arg Glu Ser Gly Ser Leu AsnPro Asp 195 200 205 His Gly Pro Ala Val Ile His Cys Ser Ala Gly Ile GlyArg Ser Gly 210 215 220 Thr Phe Ser Leu Val Asp Thr Cys Leu Val Leu MetGlu Lys Gly Glu 225 230 235 240 Asp Val Asn Val Lys Gln Ile Leu Leu SerMet Arg Lys Tyr Arg Met 245 250 255 Gly Leu Ile Gln Thr Pro Asp Gln LeuArg Phe Ser Tyr Met Ala Ile 260 265 270 Ile Glu Gly Ala Lys Tyr Thr LysGly Asp Ser Asn Ile Gln Asn Arg 275 280 285 Thr Met Thr Glu Lys Tyr AsnGly Lys Arg Ile Gly Ser Glu Asp Glu 290 295 300 Lys Leu Thr Gly Leu SerSer Lys Val Pro Asp Thr Val Glu Glu Ser 305 310 315 320 Ser Glu Ser IleLeu Arg Lys Arg Ile Arg Glu Asp Arg Lys Ala Thr 325 330 335 Thr Ala GlnLys Val Gln Gln Met Arg Gln Arg Leu Asn Glu Thr Glu 340 345 350 Arg LysArg Lys Arg Pro Arg Leu Thr Asp Thr 355 360 11 2477 DNA Homo sapiens 11gctcgggcgc cgagtctgcg cgctgacgtc cgacgctcca ggtactttcc ccacggccga 60cagggcttgg cgtgggggcg gggcgcggcg cgcagcgcgc atgcgccgca gcgccagcgc 120tctccccgga tcgtgcgggg cctgagcctc tccgccggcg caggctctgc tcgcgccagc 180tcgctcccgc agccatgccc accaccatcg agcgggagtt cgaagagttg gatactcagc 240gtcgctggca gccgctgtac ttggaaattc gaaatgagtc ccatgactat cctcatagag 300tggccaagtt tccagaaaac agaaatcgaa acagatacag agatgtaagc ccatatgatc 360acagtcgtgt taaactgcaa aatgctgaga atgattatat taatgccagt ttagttgaca 420tagaagaggc acaaaggagt tacatcttaa cacagggtcc acttcctaac acatgctgcc 480atttctggct tatggtttgg cagcagaaga ccaaagcagt tgtcatgctg aaccgcattg 540tggagaaaga atcggttaaa tgtgcacagt actggccaac agatgaccaa gagatgctgt 600ttaaagaaac aggattcagt gtgaagctct tgtcagaaga tgtgaagtcg tattatacag 660tacatctact acaattagaa aatatcaata gtggtgaaac cagaacaata tctcactttc 720attatactac ctggccagat tttggagtcc ctgaatcacc agcttcattt ctcaatttct 780tgtttaaagt gagagaatct ggctccttga accctgacca tgggcctgcg gtgatccact 840gtagtgcagg cattgggcgc tctggcacct tctctctggt agacacttgt cttgttttga 900tggaaaaagg agatgatatt aacataaaac aagtgttact gaacatgaga aaataccgaa 960tgggtcttat tcagacccca gatcaactga gattctcata catggctata atagaaggag 1020caaaatgtat aaagggagat tctagtatac agaaacgatg gaaagaactt tctaaggaag 1080acttatctcc tgcctttgat cattcaccaa acaaaataat gactgaaaaa tacaatggga 1140acagaatagg tctagaagaa gaaaaactga caggtgaccg atgtacagga ctttcctcta 1200aaatgcaaga tacaatggag gagaacagtg agagtgctct acggaaacgt attcgagagg 1260acagaaaggc caccacagct cagaaggtgc agcagatgaa acagaggcta aatgagaatg 1320aacgaaaaag aaaaaggtgg ttatattggc aacctattct cactaagatg gggtttatgt 1380cagtcatttt ggttggcgct tttgttggct ggagactgtt ttttcagcaa aatgccctat 1440aaacaattaa ttttgcccag caagcttctg cactagtaac tgacagtgct acattaatca 1500taggggtttg tctgcagcaa acgcctcata tcccaaaaac ggtgcagtag aatagacatc 1560aaccagataa gtgatattta cagtcacaag cccaacatct caggactctt gactgcaggt 1620tcctctgaac cccaaactgt aaatggctgt ctaaaataaa gacattcatg tttgttaaaa 1680actggtaaat tttgcaactg tattcataca tgtcaaacac agtatttcac ctgaccaaca 1740ttgagatatc ctttatcaca ggatttgttt ttggaggcta tctggatttt aacctgcact 1800tgatataagc aataaatatt gtggttttat ctacgttatt ggaaagaaaa tgacatttaa 1860ataatgtgtg taatgtataa tgtactattg acatgggcat caacactttt attcttaagc 1920atttcagggt aaatatattt tataagtatc tatttaatct tttgtagtta actgtacttt 1980ttaagagctc aatttgaaaa atctgttact aaaaaaaaaa attgtatgtc gattgaattg 2040tactggatac attttccatt tttctaaaaa gaagtttgat atgagcagtt agaagttgga 2100ataagcaatt tctactatat attgcatttc ttttatgttt tacagttttc cccattttaa 2160aaagaaaagc aaacaaagaa acaaaagttt ttcctaaaaa tatctttgaa ggaaaattct 2220ccttactggg atagtcaggt aaacagttgg tcaagacttt gtaaagaaat tggtttctgt 2280aaatcccatt attgatatgt ttatttttca tgaaaatttc aatgtagttg gggtagatta 2340tgatttagga agcaaaagta agaagcagca ttttatgatt cataatttca gtttactaga 2400ctgaagtttt gaagtaaaca cttttcagtt tctttctact tcaataaata gtatgattat 2460atgcaaacct taaaaaa 2477 12 415 PRT Homo sapiens 12 Met Pro Thr Thr IleGlu Arg Glu Phe Glu Glu Leu Asp Thr Gln Arg 1 5 10 15 Arg Trp Gln ProLeu Tyr Leu Glu Ile Arg Asn Glu Ser His Asp Tyr 20 25 30 Pro His Arg ValAla Lys Phe Pro Glu Asn Arg Asn Arg Asn Arg Tyr 35 40 45 Arg Asp Val SerPro Tyr Asp His Ser Arg Val Lys Leu Gln Asn Ala 50 55 60 Glu Asn Asp TyrIle Asn Ala Ser Leu Val Asp Ile Glu Glu Ala Gln 65 70 75 80 Arg Ser TyrIle Leu Thr Gln Gly Pro Leu Pro Asn Thr Cys Cys His 85 90 95 Phe Trp LeuMet Val Trp Gln Gln Lys Thr Lys Ala Val Val Met Leu 100 105 110 Asn ArgIle Val Glu Lys Glu Ser Val Lys Cys Ala Gln Tyr Trp Pro 115 120 125 ThrAsp Asp Gln Glu Met Leu Phe Lys Glu Thr Gly Phe Ser Val Lys 130 135 140Leu Leu Ser Glu Asp Val Lys Ser Tyr Tyr Thr Val His Leu Leu Gln 145 150155 160 Leu Glu Asn Ile Asn Ser Gly Glu Thr Arg Thr Ile Ser His Phe His165 170 175 Tyr Thr Thr Trp Pro Asp Phe Gly Val Pro Glu Ser Pro Ala SerPhe 180 185 190 Leu Asn Phe Leu Phe Lys Val Arg Glu Ser Gly Ser Leu AsnPro Asp 195 200 205 His Gly Pro Ala Val Ile His Cys Ser Ala Gly Ile GlyArg Ser Gly 210 215 220 Thr Phe Ser Leu Val Asp Thr Cys Leu Val Leu MetGlu Lys Gly Asp 225 230 235 240 Asp Ile Asn Ile Lys Gln Val Leu Leu AsnMet Arg Lys Tyr Arg Met 245 250 255 Gly Leu Ile Gln Thr Pro Asp Gln LeuArg Phe Ser Tyr Met Ala Ile 260 265 270 Ile Glu Gly Ala Lys Cys Ile LysGly Asp Ser Ser Ile Gln Lys Arg 275 280 285 Trp Lys Glu Leu Ser Lys GluAsp Leu Ser Pro Ala Phe Asp His Ser 290 295 300 Pro Asn Lys Ile Met ThrGlu Lys Tyr Asn Gly Asn Arg Ile Gly Leu 305 310 315 320 Glu Glu Glu LysLeu Thr Gly Asp Arg Cys Thr Gly Leu Ser Ser Lys 325 330 335 Met Gln AspThr Met Glu Glu Asn Ser Glu Ser Ala Leu Arg Lys Arg 340 345 350 Ile ArgGlu Asp Arg Lys Ala Thr Thr Ala Gln Lys Val Gln Gln Met 355 360 365 LysGln Arg Leu Asn Glu Asn Glu Arg Lys Arg Lys Arg Trp Leu Tyr 370 375 380Trp Gln Pro Ile Leu Thr Lys Met Gly Phe Met Ser Val Ile Leu Val 385 390395 400 Gly Ala Phe Val Gly Trp Arg Leu Phe Phe Gln Gln Asn Ala Leu 405410 415 13 1714 DNA Homo sapiens 13 gctcgggcgc cgagtctgcg cgctgacgtccgacgctcca ggtactttcc ccacggccga 60 cagggcttgg cgtgggggcg gggcgcggcgcgcagcgcgc atgcgccgca gcgccagcgc 120 tctccccgga tcgtgcgggg cctgagcctctccgccggcg caggctctgc tcgcgccagc 180 tcgctcccgc agccatgccc accaccatcgagcgggagtt cgaagagttg gatactcagc 240 gtcgctggca gccgctgtac ttggaaattcgaaatgagtc ccatgactat cctcatagag 300 tggccaagtt tccagaaaac agaaatcgaaacagatacag agatgtaagc ccatatgatc 360 acagtcgtgt taaactgcaa aatgctgagaatgattatat taatgccagt ttagttgaca 420 tagaagaggc acaaaggagt tacatcttaacacagggtcc acttcctaac acatgctgcc 480 atttctggct tatggtttgg cagcagaagaccaaagcagt tgtcatgctg aaccgcattg 540 tggagaaaga atcggttaaa tgtgcacagtactggccaac agatgaccaa gagatgctgt 600 ttaaagaaac aggattcagt gtgaagctcttgtcagaaga tgtgaagtcg tattatacag 660 tacatctact acaattagaa aatatcaatagtggtgaaac cagaacaata tctcactttc 720 attatactac ctggccagat tttggagtccctgaatcacc agcttcattt ctcaatttct 780 tgtttaaagt gagagaatct ggctccttgaaccctgacca tgggcctgcg gtgatccact 840 gtagtgcagg cattgggcgc tctggcaccttctctctggt agacacttgt cttgttttga 900 tggaaaaagg agatgatatt aacataaaacaagtgttact gaacatgaga aaataccgaa 960 tgggtcttat tcagacccca gatcaactgagattctcata catggctata atagaaggag 1020 caaaatgtat aaagggagat tctagtatacagaaacgatg gaaagaactt tctaaggaag 1080 acttatctcc tgcctttgat cattcaccaaacaaaataat gactgaaaaa tacaatggga 1140 acagaatagg tctagaagaa gaaaaactgacaggtgaccg atgtacagga ctttcctcta 1200 aaatgcaaga tacaatggag gagaacagtgagagtgctct acggaaacgt attcgagagg 1260 acagaaaggc caccacagct cagaaggtgcagcagatgaa acagaggcta aatgagaatg 1320 aacgaaaaag aaaaaggcca agattgacagacacctaata ttcatgactt gagaatattc 1380 tgcagctata aattttgaac cattgatgtgcaaagcaaga cctgaagccc actccggaaa 1440 ctaaagtgag gctcgctaac cctctagattgcctcacagt tgtttgttta caaagtaaac 1500 tttacatcca ggggatgaag agcacccaccagcagaagac tttgcagaac ctttaattgg 1560 atgtgttaag tgtttttaat gagtgtatgaaatgtagaaa gatgtacaag aaataaatta 1620 ggagagatta ctttgtattg tactgccattcctactgtat ttttatactt tttggcagca 1680 ttaaatattt ttgttaaata aaaaaaaaaaaaaa 1714 14 387 PRT Homo sapiens 14 Met Pro Thr Thr Ile Glu Arg Glu PheGlu Glu Leu Asp Thr Gln Arg 1 5 10 15 Arg Trp Gln Pro Leu Tyr Leu GluIle Arg Asn Glu Ser His Asp Tyr 20 25 30 Pro His Arg Val Ala Lys Phe ProGlu Asn Arg Asn Arg Asn Arg Tyr 35 40 45 Arg Asp Val Ser Pro Tyr Asp HisSer Arg Val Lys Leu Gln Asn Ala 50 55 60 Glu Asn Asp Tyr Ile Asn Ala SerLeu Val Asp Ile Glu Glu Ala Gln 65 70 75 80 Arg Ser Tyr Ile Leu Thr GlnGly Pro Leu Pro Asn Thr Cys Cys His 85 90 95 Phe Trp Leu Met Val Trp GlnGln Lys Thr Lys Ala Val Val Met Leu 100 105 110 Asn Arg Ile Val Glu LysGlu Ser Val Lys Cys Ala Gln Tyr Trp Pro 115 120 125 Thr Asp Asp Gln GluMet Leu Phe Lys Glu Thr Gly Phe Ser Val Lys 130 135 140 Leu Leu Ser GluAsp Val Lys Ser Tyr Tyr Thr Val His Leu Leu Gln 145 150 155 160 Leu GluAsn Ile Asn Ser Gly Glu Thr Arg Thr Ile Ser His Phe His 165 170 175 TyrThr Thr Trp Pro Asp Phe Gly Val Pro Glu Ser Pro Ala Ser Phe 180 185 190Leu Asn Phe Leu Phe Lys Val Arg Glu Ser Gly Ser Leu Asn Pro Asp 195 200205 His Gly Pro Ala Val Ile His Cys Ser Ala Gly Ile Gly Arg Ser Gly 210215 220 Thr Phe Ser Leu Val Asp Thr Cys Leu Val Leu Met Glu Lys Gly Asp225 230 235 240 Asp Ile Asn Ile Lys Gln Val Leu Leu Asn Met Arg Lys TyrArg Met 245 250 255 Gly Leu Ile Gln Thr Pro Asp Gln Leu Arg Phe Ser TyrMet Ala Ile 260 265 270 Ile Glu Gly Ala Lys Cys Ile Lys Gly Asp Ser SerIle Gln Lys Arg 275 280 285 Trp Lys Glu Leu Ser Lys Glu Asp Leu Ser ProAla Phe Asp His Ser 290 295 300 Pro Asn Lys Ile Met Thr Glu Lys Tyr AsnGly Asn Arg Ile Gly Leu 305 310 315 320 Glu Glu Glu Lys Leu Thr Gly AspArg Cys Thr Gly Leu Ser Ser Lys 325 330 335 Met Gln Asp Thr Met Glu GluAsn Ser Glu Ser Ala Leu Arg Lys Arg 340 345 350 Ile Arg Glu Asp Arg LysAla Thr Thr Ala Gln Lys Val Gln Gln Met 355 360 365 Lys Gln Arg Leu AsnGlu Asn Glu Arg Lys Arg Lys Arg Pro Arg Leu 370 375 380 Thr Asp Thr 38515 21 RNA Artificial Sequence Small interfering RNA - mTCPTP1.1 15guugucaugc uaaaccgaac n 21 16 19 RNA Artificial Sequence Smallinterfering RNA - mTCPTP1.1 16 guugucaugc uaaaccgaa 19 17 19 RNAArtificial Sequence Small interfering RNA - mTCPTP1.1 17 uucgguuuagcaugacaac 19 18 21 RNA Artificial Sequence Small interfering RNA -mTCPTP1.1 18 guugucaugc uaaaccgaan n 21 19 21 RNA Artificial SequenceSmall interfering RNA - mTCPTP1.1 19 nnuucgguuu agcaugacaa c 21 20 21RNA Artificial Sequence Small interfering RNA - mTCPTP1.2 20 cagaacagagugaugguuga g 21 21 19 RNA Artificial Sequence Small interfering RNA -mTCPTP1.2 21 cagaacagag ugaugguug 19 22 19 RNA Artificial Sequence Smallinterfering RNA - mTCPTP1.2 22 caaccaucac ucuguucug 19 23 21 RNAArtificial Sequence Small interfering RNA - mTCPTP1.2 23 cagaacagagugaugguugn n 21 24 21 RNA Artificial Sequence Small interfering RNA -mTCPTP1.2 24 nncaaccauc acucuguucu g 21 25 36 DNA Artificial SequenceOligonucleotide primer (TC45 5′ BamHI) 25 ggggggatcc atgcccaccaccatcgagcg ggagtt 36 26 49 DNA Artificial Sequence Oligonucleotideprimer (TC45 3′ EcoRI) 26 ggggaattct taggtgtctg tcaatcttgg cctttttctttttcgttca 49 27 21 RNA Artificial Sequence Small interfering RNA -hTCPTP1.4 27 guugucaugc ugaaccgcan n 21 28 19 RNA Artificial SequenceSmall interfering RNA - hTCPTP1.4 28 guugucaugc ugaaccgca 19 29 19 RNAArtificial Sequence Small interfering RNA - hTCPTP1.4 29 ugcgguucagcaugacaac 19 30 21 RNA Artificial Sequence Small interfering RNA -hTCPTP1.4 30 guugucaugc ugaaccgcan n 21 31 21 RNA Artificial SequenceSmall interfering RNA - hTCPTP1.4 31 nnugcgguuc agcaugacaa c 21 32 21RNA Artificial Sequence Small interfering RNA - hTCPTP1.5 32 gcccauaugaucacagucgn g 21 33 19 RNA Artificial Sequence Small interfering RNA -hTCPTP1.5 33 gcccauauga ucacagucg 19 34 19 RNA Artificial Sequence Smallinterfering RNA - hTCPTP1.5 34 cgacugugau cauaugggc 19 35 21 RNAArtificial Sequence Small interfering RNA - hTCPTP1.5 35 gcccauaugaucacagucgn n 21 36 21 RNA Artificial Sequence Small interfering RNA -hTCPTP1.5 36 nncgacugug aucauauggg c 21 37 21 RNA Artificial SequenceSmall interfering RNA - hTCPTP1.6 37 ucgguuaaau gugcacagua c 21 38 19RNA Artificial Sequence Small interfering RNA - hTCPTP1.6 38 ucgguuaaaugugcacagu 19 39 19 RNA Artificial Sequence Small interfering RNA -hTCPTP1.6 39 acugugcaca uuuaaccga 19 40 21 RNA Artificial Sequence Smallinterfering RNA - hTCPTP1.6 40 ucgguuaaau gugcacagun n 21 41 21 RNAArtificial Sequence Small interfering RNA - hTCPTP1.6 41 nnacugugcacauuuaaccg a 21 42 21 RNA Artificial Sequence Small interfering RNA -hTCPTP1.7 42 ugacuauccu cauagagugg g 21 43 19 RNA Artificial SequenceSmall interfering RNA - hTCPTP1.7 43 ugacuauccu cauagagug 19 44 19 RNAArtificial Sequence Small interfering RNA - hTCPTP1.7 44 cacucuaugaggauaguca 19 45 21 RNA Artificial Sequence Small interfering RNA -hTCPTP1.7 45 ugacuauccu cauagagugn n 21 46 21 RNA Artificial SequenceSmall interfering RNA - hTCPTP1.7 46 nncacucuau gaggauaguc a 21 47 21RNA Artificial Sequence Small interfering RNA - hTCPTP1.1 47 agugagagaaucuggcuccn n 21 48 19 RNA Artificial Sequence Small interfering RNA -hTCPTP1.1 48 agugagagaa ucuggcucc 19 49 19 RNA Artificial Sequence Smallinterfering RNA - hTCPTP1.1 49 ggagccagau ucucucacu 19 50 21 RNAArtificial Sequence Small interfering RNA - hTCPTP1.1 50 agugagagaaucuggcuccn n 21 51 21 RNA Artificial Sequence Small interfering RNA -hTCPTP1.1 51 nnggagccag auucucucac u 21 52 19 RNA Artificial SequenceSmall interfering RNA - hTCPTP1.2 52 ggaagacuua ucuccugcc 19 53 19 RNAArtificial Sequence Small interfering RNA - hTCPTP1.2 53 ggaagacuuaucuccugcc 19 54 19 RNA Artificial Sequence Small interfering RNA -hTCPTP1.2 54 ggcaggagau aagucuucc 19 55 21 RNA Artificial Sequence Smallinterfering RNA - hTCPTP1.2 55 ggaagacuua ucuccugccn n 21 56 21 RNAArtificial Sequence Small interfering RNA - hTCPTP1.2 56 nnggcaggagauaagucuuc c 21 57 21 RNA Artificial Sequence Small interfering RNA -hTCPTP1.3 57 ggugaccgau guacaggacn n 21 58 19 RNA Artificial SequenceSmall interfering RNA - hTCPTP1.3 58 ggugaccgau guacaggac 19 59 19 RNAArtificial Sequence Small interfering RNA - hTCPTP1.3 59 guccuguacaucggucacc 19 60 21 RNA Artificial Sequence Small interfering RNA -hTCPTP1.3 60 ggugaccgau guacaggacn n 21 61 21 RNA Artificial SequenceSmall interfering RNA - hTCPTP1.3 61 nnguccugua caucggucac c 21 62 21RNA Artificial Sequence Small interfering RNA - rPTP1B1.2 62 cggaugguggguggaggucn n 21 63 19 RNA Artificial Sequence Small interfering RNA -rPTP1B1.2 63 cggauggugg guggagguc 19 64 19 RNA Artificial Sequence Smallinterfering RNA - rPTP1B1.2 64 gaccuccacc caccauccg 19 65 21 RNAArtificial Sequence Small interfering RNA - rPTP1B1.2 65 cggaugguggguggaggucn n 21 66 21 RNA Artificial Sequence Small interfering RNA -rPTP1B1.2 66 nngaccucca cccaccaucc g 21 67 21 DNA Artificial SequenceSmall interefering RNA - TCPTP1 67 aacagauaca gagauguaag c 21 68 19 RNAArtificial Sequence Small interefering RNA - TCPTP1 68 aacagauacagagauguaa 19 69 19 RNA Artificial Sequence Small interefering RNA -TCPTP1 69 uuacaucucu guaucuguu 19 70 21 RNA Artificial Sequence Smallinterefering RNA - TCPTP1 70 aacagauaca gagauguaan n 21 71 21 RNAArtificial Sequence Small interefering RNA - TCPTP1 71 nnuuacaucucuguaucugu u 21 72 21 RNA Artificial Sequence Small interefering RNA -TCPTP2 72 aagcccauau gaucacaguc g 21 73 19 RNA Artificial Sequence Smallinterefering RNA - TCPTP2 73 aagcccauau gaucacagu 19 74 19 RNAArtificial Sequence Small interefering RNA - TCPTP2 74 acugugaucauaugggcuu 19 75 21 RNA Artificial Sequence Small interefering RNA -TCPTP2 75 aagcccauau gaucacagun n 21 76 21 RNA Artificial Sequence Smallinterefering RNA - TCPTP2 76 nnacugugau cauaugggcu u 21

What is claimed is:
 1. An isolated small interfering RNA (siRNA)polynucleotide, comprising at least one nucleotide sequence selectedfrom the group consisting of SEQ ID NOS:28-31, 33-36, 38-41, and 68-71.2. The small interfering RNA polynucleotide of claim 1 that comprises atleast one nucleotide sequence selected from the group consisting of SEQID NOS: 28-31, 33-36, 38-41, and 68-71 and the complementarypolynucleotide thereto.
 3. A small interfering RNA polynucleotide ofeither claim 1 or claim 2 that is capable of interfering with expressionof a TCPTP polypeptide, wherein the TCPTP polypeptide comprises an aminoacid sequence as set forth in a sequence selected from the groupconsisting of GenBank Acc. Nos. M25393, NM_(—)002828, NM_(—)080422, andSEQ ID NOS:4, 12, and
 14. 4. The siRNA polynucleotide of either claim 1or claim 2 wherein the nucleotide sequence of the siRNA polynucleotidediffers by one, two, three or four nucleotides at any of positions 1-19of a sequence selected from the group consisting of the sequences setforth in SEQ ID NOS: 28-31, 33-36, 38-41, and 68-71.
 5. The siRNApolynucleotide of either claim 1 or claim 2 wherein the nucleotidesequence of the siRNA polynucleotide differs by at least two, three orfour nucleotides at any of positions 1-19 of a sequence selected fromthe group consisting of the sequences set forth in SEQ ID NOS: 28-31,33-36, 38-41, and 68-71.
 6. An isolated siRNA polynucleotide comprisinga nucleotide sequence according to SEQ ID NO: 28, or the complementthereof.
 7. An isolated siRNA polynucleotide comprising a nucleotidesequence according to SEQ ID NO: 33, or the complement thereof.
 8. Anisolated siRNA polynucleotide comprising a nucleotide sequence accordingto SEQ ID NO: 38, or the complement thereof.
 9. An isolated siRNApolynucleotide comprising a nucleotide sequence according to SEQ ID NO:68, or the complement thereof.
 10. The siRNA polynucleotide of claim 1or claim 2 wherein the polynucleotide comprises at least one syntheticnucleotide analogue of a naturally occurring nucleotide.
 11. The siRNApolynucleotide of claim 1 or claim 2 wherein the polynucleotide islinked to a detectable label.
 12. The siRNA polynucleotide of claim 11wherein the detectable label is a reporter molecule.
 13. The siRNA ofclaim 12 wherein the reporter molecule is selected from the groupconsisting of a dye, a radionuclide, a luminescent group, a fluorescentgroup, and biotin.
 14. The siRNA polynucleotide of claim 13 wherein thefluorescent group is fluorescein isothiocyanate.
 15. The siRNApolynucleotide of claim 11 wherein the detectable label is a magneticparticle.
 16. A pharmaceutical composition comprising the siRNApolynucleotide of either claim 1 or claim 2 and a physiologicallyacceptable carrier.
 17. The pharmaceutical composition of claim 16wherein the carrier comprises a liposome.
 18. A recombinant nucleic acidconstruct comprising a polynucleotide that is capable of directingtranscription of a small interfering RNA (siRNA), the polynucleotidecomprising: (i) a first promoter; (ii) a second promoter; and (iii) atleast one DNA polynucleotide segment comprising at least one nucleotidesequence selected from the group consisting of SEQ ID NOS: 28-31, 33-36,38-41, and 68-71, or a complement thereto, wherein each DNApolynucleotide segment and its complement are operably linked to atleast one of the first and second promoters, and wherein the promotersare oriented to direct transcription of the DNA polynucleotide segmentand its reverse complement.
 19. The recombinant nucleic acid constructof claim 18, comprising at least one enhancer that is selected from afirst enhancer operably linked to the first promoter and a secondenhancer operably linked to the second promoter.
 20. The recombinantnucleic acid construct of claim 18, comprising at least onetranscriptional terminator that is selected from (i) a firsttranscriptional terminator that is positioned in the construct toterminate transcription directed by the first promoter and (ii) a secondtranscriptional terminator that is positioned in the construct toterminate transcription directed by the second promoter.
 21. Therecombinant nucleic acid construct of claim 18 wherein the siRNA iscapable of interfering with expression of a TCPTP polypeptide, whereinthe TCPTP polypeptide comprises an amino acid sequence as set forth in asequence selected from the group consisting of GenBank Acc. Nos. M25393,NM_(—)002828, and NM_(—)080422, SEQ ID NOS:4, 12, and
 14. 22. Arecombinant nucleic acid construct comprising a polynucleotide that iscapable of directing transcription of a small interfering RNA (siRNA),the polynucleotide comprising at least one promoter and a DNApolynucleotide segment, wherein the DNA polynucleotide segment isoperably linked to the promoter, and wherein the DNA polynucleotidesegment comprises (i) at least one DNA polynucleotide that comprises atleast one nucleotide sequence selected from the group consisting of SEQID NOS: 28-31, 33-36, 38-41, and 68-71, or a complement thereto; (ii) aspacer sequence comprising at least 4 nucleotides operably linked to theDNA polynucleotide of (i); and (iii) the reverse complement of the DNApolynucleotide of (i) operably linked to the spacer sequence.
 23. Therecombinant nucleic acid construct of claim 22 wherein the siRNAcomprises an overhang of at least one and no more than four nucleotides,the overhang being located immediately 3′ to (iii).
 24. The recombinantnucleic acid construct of claim 22 wherein the spacer sequence comprisesat least 9 nucleotides.
 25. The recombinant nucleic acid construct ofclaim 22 wherein the spacer sequence comprises two uridine nucleotidesthat are contiguous with (iii).
 26. The recombinant nucleic acidconstruct of claim 22 comprising at least one transcriptional terminatorthat is operably linked to the DNA polynucleotide segment.
 27. A hostcell transformed or transfected with the recombinant nucleic acidconstruct of any one of claims 18-26.
 28. A pharmaceutical compositioncomprising an siRNA polynucleotide and a physiologically acceptablecarrier, wherein the siRNA polynucleotide is selected from the groupconsisting of: (i) an RNA polynucleotide which comprises at least onenucleotide sequence selected from the group consisting of SEQ ID NOS:28-31, 33-36, 38-41, and 68-71, (ii) an RNA polynucleotide thatcomprises at least one nucleotide sequence selected from the groupconsisting of SEQ ID NOS: 28-31, 33-36, 38-41, and 68-71and thecomplementary polynucleotide thereto, (iii) an RNA polynucleotideaccording to (i) or (ii) wherein the nucleotide sequence of the siRNApolynucleotide differs by one, two or three nucleotides at any ofpositions 1-19 of a sequence selected from the group consisting of thesequences set forth in SEQ ID NOS: 28-31, 33-36, 38-41, and 68-71, and(iv) an RNA polynucleotide according to (i) or (ii) wherein thenucleotide sequence of the siRNA polynucleotide differs by two, three orfour nucleotides at any of positions 1-19 of a sequence selected fromthe group consisting of the sequences set forth in SEQ ID NOS: 28-31,33-36, 38-41, and 68-71.
 29. The pharmaceutical composition of claim 28wherein the carrier comprises a liposome.
 30. A method for interferingwith expression of a TCPTP polypeptide, or variant thereof, comprisingcontacting a subject that comprises at least one cell which is capableof expressing a TCPTP polypeptide with a siRNA polynucleotide for a timeand under conditions sufficient to interfere with TCPTP polypeptideexpression, wherein: (a) the PTP1B polypeptide comprises an amino acidsequence as set forth in a sequence selected from the group consistingof GenBank Acc. Nos. M25393, NM_(—)002828, and NM_(—)080422, SEQ IDNOS:4, 12, and 14, (b) the siRNA polynucleotide is selected from thegroup consisting of (i) an RNA polynucleotide which comprises at leastone nucleotide sequence selected from the group consisting of SEQ IDNOS: 28-31, 33-36, 38-41, and 68-71, (ii) an RNA polynucleotide thatcomprises at least one nucleotide sequence selected from the groupconsisting of SEQ ID NOS: 28-31, 33-36, 38-41, and 68-71and thecomplementary polynucleotide thereto, (iii) an RNA polynucleotideaccording to (i) or (ii) wherein the nucleotide sequence of the siRNApolynucleotide differs by one, two or three nucleotides at any ofpositions 1-19 of a sequence selected from the group consisting of thesequences set forth in SEQ ID NOS: 28-31, 33-36, 38-41, and 68-71, and(iv) an RNA polynucleotide according to (i) or (ii) wherein thenucleotide sequence of the siRNA polynucleotide differs by two, three orfour nucleotides at any of positions 1-19 of a sequence selected fromthe group consisting of the sequences set forth in SEQ ID NOS: 28-31,33-36, 38-41, and 68-71.
 31. A method for interfering with expression ofa TCPTP polypeptide that comprises an amino acid sequence as set forthin a sequence selected from the group consisting of GenBank Acc. Nos.M25393, NM_(—)002828, and NM_(—)080422, SEQ ID NOS:4, 12, and 14, or avariant of said TCPTP polypeptide, said method comprising contacting,under conditions and for a time sufficient to interfere with TCPTPpolypeptide expression, (i) a subject that comprises at least one cellthat is capable of expressing the TCPTP polypeptide, and (ii) arecombinant nucleic acid construct according to either claim 18 or claim22.
 32. A method for identifying a component of a TCPTP signaltransduction pathway comprising: A. contacting a siRNA polynucleotideand a first biological sample comprising at least one cell that iscapable of expressing a TCPTP polypeptide, or a variant of said TCPTPpolypeptide, under conditions and for a time sufficient for TCPTPexpression when the siRNA polynucleotide is not present, wherein (1) theTCPTP polypeptide comprises an amino acid sequence as set forth in asequence selected from the group consisting of GenBank Acc. Nos. M25393,NM_(—)002828, and NM_(—)080422, SEQ ID NOS:4, 12, and 14, (2) the siRNApolynucleotide is selected from the group consisting of (i) an RNApolynucleotide which comprises at least one nucleotide sequence selectedfrom the group consisting of SEQ ID NOS: 28-31, 33-36, 38-41, and 68-71,(ii) an RNA polynucleotide that comprises at least one nucleotidesequence selected from the group consisting of SEQ ID NOS: 28-31, 33-36,38-41, and 68-71and the complementary polynucleotide thereto, (iii) anRNA polynucleotide according to (i) or (ii) wherein the nucleotidesequence of the siRNA polynucleotide differs by one, two or threenucleotides at any of positions 1-19 of a sequence selected from thegroup consisting of the sequences set forth in SEQ ID NOS: 28-31, 33-36,38-41, and 68-71, and (iv) an RNA polynucleotide according to (i) or(ii) wherein the nucleotide sequence of the siRNA polynucleotide differsby two, three or four nucleotides at any of positions 1-19 of a sequenceselected from the group consisting of the sequences set forth in SEQ IDNOS: 28-31, 33-36, 38-41, and 68-71; and B. comparing a level ofphosphorylation of at least one protein that is capable of beingphosphorylated in the cell with a level of phosphorylation of theprotein in a control sample that has not been contacted with the siRNApolynucleotide, wherein an altered level of phosphorylation of theprotein in the presence of the siRNA polynucleotide relative to thelevel of phosphorylation of the protein in an absence of the siRNApolynucleotide indicates that the protein is a component of the TCPTPsignal transduction pathway.
 33. The method of claim 32 wherein thesignal transduction pathway comprises a Jak2 kinase.
 34. A method formodulating an insulin receptor protein phosphorylation state in a cell,comprising contacting the cell with a siRNA polynucleotide underconditions and for a time sufficient to interfere with expression of aTCPTP polypeptide, wherein (a) the TCPTP polypeptide comprises an aminoacid sequence as set forth in a sequence selected from the groupconsisting of GenBank Acc. Nos. M25393, NM_(—)002828, and NM_(—)080422,SEQ ID NOS:4, 12, and 14, (b) the siRNA polynucleotide is selected fromthe group consisting of (i) an RNA polynucleotide which comprises atleast one nucleotide sequence selected from the group consisting of SEQID NOS: 28-31, 33-36, 38-41, and 68-71, or the complements thereof, (ii)an RNA polynucleotide that comprises at least one nucleotide sequenceselected from the group consisting of SEQ ID NOS: 28-31, 33-36, 38-41,and 68-71and the complementary polynucleotide thereto, (iii) an RNApolynucleotide according to (i) or (ii) wherein the nucleotide sequenceof the siRNA polynucleotide differs by one, two or three nucleotides atany of positions 1-19 of a sequence selected from the group consistingof the sequences set forth in SEQ ID NOS: 28-31, 33-36, 38-41, and68-71, and (iv) an RNA polynucleotide according to (i) or (ii) whereinthe nucleotide sequence of the siRNA polynucleotide differs by two,three or four nucleotides at any of positions 1-19 of a sequenceselected from the group consisting of the sequences set forth in SEQ IDNOS: 28-31, 33-36, 38-41, and 68-71; and (c) the insulin receptorprotein comprises a polypeptide which comprises an amino acid sequenceselected from the group consisting of SEQ ID NOS:______-______, or avariant thereof.
 35. A method for altering Jak2 protein phosphorylationstate in a cell, comprising contacting the cell with a siRNApolynucleotide under conditions and for a time sufficient to interferewith expression of a TCPTP polypeptide, wherein (a) the TCPTPpolypeptide comprises an amino acid sequence as set forth in a sequenceselected from the group consisting of GenBank Acc. Nos. M25393,NM_(—)002828, and NM_(—)080422, SEQ ID NOS:4, 12, and 14, (b) the siRNApolynucleotide is selected from the group consisting of (i) an RNApolynucleotide which comprises at least one nucleotide sequence selectedfrom the group consisting of SEQ ID NOS: 28-31, 33-36, 38-41, and 68-71,or the complements thereof, (ii) an RNA polynucleotide that comprises atleast one nucleotide sequence selected from the group consisting of SEQID NOS: 28-31, 33-36, 38-41, and 68-71and the complementarypolynucleotide thereto, (iii) an RNA polynucleotide according to (i) or(ii) wherein the nucleotide sequence of the siRNA polynucleotide differsby one, two or three nucleotides at any of positions 1-19 of a sequenceselected from the group consisting of the sequences set forth in SEQ IDNOS: 28-31, 33-36, 38-41, and 68-71, and (iv) an RNA polynucleotideaccording to (i) or (ii) wherein the nucleotide sequence of the siRNApolynucleotide differs by two, three or four nucleotides at any ofpositions 1-19 of a sequence selected from the group consisting of thesequences set forth in SEQ ID NOS: 28-31, 33-36, 38-41, and 68-71; and(c) the Jak2 protein comprises a polypeptide which comprises an aminoacid sequence selected from the group consisting of SEQ IDNOS:______-______, or a variant thereof.
 36. A method for treating aJak2-associated disorder comprising administering to a subject in needthereof a pharmaceutical composition according to claim 28, wherein thesiRNA polynucleotide inhibits expression of a TCPTP polypeptide, or avariant thereof.
 37. The method of claim 36 wherein the Jak2-associateddisorder is selected from the group consisting of diabetes, obesity,hyperglycemia-induced apoptosis, inflammation, and a neurodegenerativedisorder.